help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2007-0334
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grant, M.
Right arrow Articles by Kumar, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grant, M.
Right arrow Articles by Kumar, U.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
Molecular Endocrinology 22 (10): 2278-2292
Copyright © 2008 by The Endocrine Society

Cell Growth Inhibition and Functioning of Human Somatostatin Receptor Type 2 Are Modulated by Receptor Heterodimerization

Michael Grant, Haydar Alturaihi, Philippe Jaquet, Brian Collier and Ujendra Kumar

Fraser Laboratories For Diabetes Research (M.G., H.A., U.K.), Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1; Department of Pharmacology and Therapeutics (M.G., B.C.), McGill University, Montreal, Quebec, Canada H3A 2K6; Faculty of Medicine (P.J.), Centre Hospitalier Universitaire Timone, 13385 Marseille, France; and Faculty of Pharmaceutical Sciences (U.K.), Division of Pharmacology and Toxicology, University of British Columbia, Vancouver, Canada V6T 1RZ

Address all correspondence and requests for reprints to: Dr. Ujendra Kumar, Faculty of Pharmaceutical Sciences, Division of Pharmacology and Toxicology, The University of British Columbia, 2146 East Mall, Vancouver, Canada V6T 1RZ. E-mail: ujkumar{at}interchange.ubc.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatostatin (SST) analogs have been successfully used in the medical treatment of acromegaly, caused by GH hypersecreting pituitary adenomas. Patients on SST analogs rarely develop tachyphylaxis despite years of continuous administration. It has been recently proposed that a functional association between SST receptor (SSTR) subtypes 2 and 5 exists to account for this behavior; however, a physical interaction has yet to be identified. Using both coimmunoprecipitation and photobleaching fluorescence resonance energy transfer microscopy techniques, we determined that SSTR2 and SSTR5 heterodimerize. Surprisingly, selective activation of SSTR2 and not SSTR5, or their costimulation, modulates the association. The SSTR2-selective agonist L-779,976 is more efficacious at inhibiting adenylate cyclase, activating ERK1/2, and inducing the cyclin-dependent kinase inhibitor p27Kip1 in cells expressing both SSTR2 and SSTR5 compared with SSTR2 alone. Furthermore, cell growth inhibition by L-779,976 treatment was markedly extended in coexpressing cells. Trafficking of SSTR2 is also affected upon heterodimerization, an attribute corresponding to modifications in β-arrestin association kinetics. Activation of SSTR2 results in the recruitment and stable association of β-arrestin, followed by receptor internalization and intracellular receptor pooling. In contrast, heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with β-arrestin. Given that SST analogs show preferential binding to SSTR2, these data provide a mechanism for their effectiveness in controlling pituitary tumors and the absence of tolerance seen in patients undergoing long-term administration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SOMATOSTATIN (SST) IS a peptide hormone that was originally identified in the hypothalamus and subsequently found throughout the central nervous system and in various peripheral organs (1). Generally classified as an inhibitory peptide, SST is secreted by endocrine, neuronal, and immune cells and acts to regulate cell secretion, neurotransmission, and cell proliferation. The physiological role of hypothalamic SST on the pituitary is well established. SST inhibits the basal and stimulated release of GH and TSH, including the secretions of prolactin and ACTH (1). SST activity is mediated by five specific receptor subtypes (SSTR1–5) that are differentially expressed in a tissue-specific manner, often with overlapping patterns of distribution (1, 2). All SSTRs possess seven transmembrane-spanning domains and are linked to G proteins, therefore belonging to the superfamily of G protein-coupled receptors (GPCRs) (1, 2). Many tumors have been shown to express SSTRs, the highest density of which is seen in tumors of neuroendocrine origin (3).

SST analogs such as lanreotide and octreotide are frequently administered as first-line treatment in acromegaly caused by GH hypersecreting pituitary adenomas to regulate endocrine function (4). Over 90% of patients on SST analogs show decreases in circulating GH levels, whereas approximately 70% of those achieve biochemical normalization. In addition, SST analog therapy frequently results in tumor shrinkage in roughly 50% of patients (3, 5, 6, 7, 8, 9). Surprisingly, patients rarely show desensitization to treatment despite years of continuous administration, a property not shared with treatment of other endocrine tumors (10). The mechanisms underlying this discrepancy are poorly understood; however, a functional association between SSTR2 and SSTR5, the primary SSTRs expressed in GH-secreting pituitary adenomas (11, 12), has been proposed to account for these actions, yet a direct physical interaction remains to be identified (13).

There is a preponderance of evidence suggesting the significance of GPCR dimerization in receptor biogenesis, regulation, and pharmacology (14, 15). Moreover, dimerization of GPCRs has been identified in various pathological states, suggesting clinical importance for such protein-protein interactions (16, 17). We have previously reported that human SSTRs can form both homo- and heterodimers. In our investigations, SSTR5 was shown to homo- and heterodimerize with SSTR1, a property that regulated receptor internalization and signaling (18, 19, 20). Furthermore, we have demonstrated that SSTR2 homodimers dissociate into monomers before internalization, an aspect that when prevented, altered the internalization rate of the receptor (21).

In the present study, we demonstrate the existence of SSTR2/SSTR5 heterodimers using coimmunoprecipitation and photobleaching fluorescence resonance energy transfer (pbFRET) microscopy techniques, an occurrence that is further augmented by selective activation of SSTR2 and not SSTR5 or their concurrent stimulation. Heterodimerization alters the association kinetics of β-arrestin to SSTR2 and augments receptor recycling. In addition, increases in the efficiency at inhibiting adenylate cyclase, activation of MAPKs, and up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 are all features observed after heterodimerization. Finally, the enhanced properties of the heterodimer conferred an extended growth-inhibitory response. Taken together, these data provide a mechanism for the effectiveness of currently available SST analogs in treating GH-secreting pituitary adenomas, given their preferential binding to SSTR2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Agonist-Bound SSTR2 Modulates Heterodimerization
To determine whether SSTR2 and SSTR5 interact to form heterodimers, we selected a HEK 293 cell clone stably coexpressing a hemagglutinin (HA)-tagged SSTR2 and a c-Myc-tagged SSTR5 for a total expression of 281 ±12 fmol/mg protein. This clone was chosen for its relatively low expression levels comparable with physiological conditions (18) and its approximate 1:1 receptor ratio (SSTR2, 167 ± 3 fmol/mg protein; SSTR5, 115 ± 2 fmol/mg protein). Heterodimerization was verified by coimmunoprecipitating SSTR2 from membrane proteins using an anti-HA antibody and immunoblotting for SSTR5 with an anti-c-Myc antibody. Under these conditions, a band estimated at 105 kDa was observed and could be identified in the absence of agonist treatment (Fig. 1BGo). The size of the heterodimeric band closely approximated the summation of the molecular weights of the monomeric species of SSTR2 and SSTR5, individually expressed in HEK 293 cells (Fig. 1AGo). We have previously demonstrated the effect of agonist on the stabilization of the SSTR1 and SSTR5 heterodimer, a property that was associated with enhanced signaling. To ascertain whether a similar mechanism was involved in the formation of the SSTR2/SSTR5 heterodimer, we selectively activated SSTR2, SSTR5, or both receptor subtypes before coimmunoprecipitation. Treatment with the SSTR2-selective agonist L-779,976 but not the SSTR5-selective agonist L-817,818 resulted in a dose-dependent increase in the formation of the heterodimer, a result that was significantly greater than basal as determined by densitometry (Fig. 1Go, B and C). Surprisingly, stimulation of both receptor subtypes with the endogenous pan-agonist SST-14 did not reveal increases in heterodimerization despite activation of SSTR2 (Fig. 1BGo). The specificity of the heterodimeric band upon coimmunoprecipitation was confirmed using the reverse combination of antibodies, whereby SSTR5 was immunoprecipitated followed by immunoblotting of SSTR2 (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).


Figure 1
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 1. Modulation of SSTR Heterodimerization by Agonist

A, Western blots of membrane proteins (50 µg) from HEK 293 cells stably expressing SSTR5 (lane 1) and SSTR2 (lane 2). B, Coimmunoprecipitation of membranes from HEK 293 cells coexpressing SSTR2 and SSTR5 treated with various concentrations of the indicated agonist before immunoprecipitation with anti-HA antibody. Membrane lysates were subjected to Western blotting with anti-c-Myc antibody. A band of approximately 105 kDa appeared as the heterodimer. C, Densitometric analysis on immunoblots in B treated with L-779,976 showing significant increases in heterodimerization. Data were analyzed using ANOVA and post hoc Dunnett’s to compare against basal. D, pbFRET microscopy on HEK 293 cells stably expressing both SSTR2 and SSTR5 after treatment with 10 nM of the indicated agonists. FRET efficiencies were analyzed using ANOVA and post hoc Dunnett’s and compared with control. E, Coexpressing cells treated with various concentrations of SST-14 and processed for pbFRET microscopy using anti-Myc monoclonal antibodies conjugated to either fluorescein (donor) or rhodamine (acceptor) to measure for SSTR5 dimerization only. Data were analyzed using ANOVA and post hoc Dunnett’s to compare against basal. Immunoblots and means ± SEM are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
To determine whether heterodimerization of SSTR2 and SSTR5 was an artifact of immunoprecipitation rather than a selective interaction occurring at the cell surface of intact cells, we performed pbFRET microscopy (20, 21, 22). Using an anti-HA antibody conjugated to fluorescein as the donor and an anti-c-Myc antibody conjugated to rhodamine as the acceptor, we probed the surface of our stable cell line for SSTR2 and SSTR5, respectively (Fig. 2Go). Under basal conditions, there was a small but sustained FRET efficiency, accordant with the coimmunoprecipitation data, characteristic of preassembled receptor complexes. Concordantly, treatment of cells with 10 nM of the SSTR2-selective compound L-779,976 resulted in a significant increase in FRET efficiency over basal that was not apparent with the addition of SST-14 or the SSTR5-selective compound L-817,818 (Fig. 1DGo). Although heterodimerization between SSTR2 and SSTR5 was unaltered after stimulation with SST-14, homodimerization of SSTR5 was observed in a dose-dependent manner (Fig. 1EGo). Taken together, these data indicate the role of ligand-bound SSTR2 in modulating the heterodimer.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 2. pbFRET Microscopy on HEK 293 Cells Stably Coexpressing SSTR2 and SSTR5

A, Confocal microscopic images illustrating SSTR2 using monoclonal anti-HA antibody conjugated to fluorescein isothiocyanate/donor (green) and SSTR5 using rabbit anti-c-Myc antibody conjugated to rhodamine/acceptor (red) and their colocalization (yellow) in HEK 293 cells. B, A representation of pbFRET microscopy on HEK 293 cells treated with 10 nM L-779,976. A selection of photobleaching micrographs taken from cells incubated with donor antibody alone under constant illumination of 488 nm light is shown. Below, a representative histogram time constant plot calculated from a portion of cell membrane on a pixel-by-pixel basis taken from approximately forty cells. The mean time constant is shown in black calculated from a Gaussian distribution curve (18.5 sec). C, Photobleaching micrographs from cells incubated with both donor and acceptor antibodies showing increases in the photobleaching time constant (23.2 sec).

 
Heterodimerization Alters the Interaction of β-Arrestin to SSTR2
A basic tenet after activation of GPCRs is their eventual desensitization and internalization (23). There have been several instances where the heterodimerization of GPCRs has altered the desensitization and internalization profiles of one or both protomers involved (15). To determine whether formation of the SSTR2/SSTR5 heterodimer altered receptor internalization, we first monitored for changes in β-arrestin interaction, a class of proteins known for their involvement in the internalization of GPCRs (24). Although both receptors, SSTR2 and SSTR5, internalize after stimulation with agonist, only SSTR2 was found to recruit β-arrestin (green) to the cell surface of HEK 293 cells (Fig. 3AGo). The high affinity of β-arrestin to SSTR2 was observed subsequent to acute treatment with both agonists SST-14 and L-779,976 and persisted in the form of endosomes after 20 min stimulation, consistent with previously reported data (25). When cotransfected cells were treated with SST-14, β-arrestin translocated from the cytosol to the cell surface within minutes of agonist exposure. However, when the cells were stimulated with agonist for a prolonged period, β-arrestin aggregated in the form of stable endosomal complexes (Fig. 3BGo). Surprisingly, when cotransfected cells were subjected to L-779,976, β-arrestin did indeed concentrate at the cell surface; nevertheless, it did not sequester in the form of endosomes after extended stimulation. This would indicate that heterodimerization interferes with β-arrestin/receptor interaction, causing uncoupling soon after the receptor internalizes.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. Differential Trafficking of β-Arrestin by SSTR2 and SSTR5

Confocal microscopy of HEK 293 cells transiently transfected with 1 µg SSTR2 or SSTR5 (A) or a combination of each receptor (B), 0.25 µg β-arrestin2-GFP, and 0.5 µg GRK2. Cells were treated with 10 nM of each agonist for the indicated times 48 h after transfection. Scale bar, 10 µm.

 
The transient interaction of β-arrestin on heterodimerization was elaborated further by confocal studies shown in Fig. 4AGo. When cotransfected cells were treated with L-779,976, β-arrestin and SSTR2 were found to colocalize within 5 min of activation but not after a prolonged stimulus despite SSTR2 internalization. However, β-arrestin was found localized with SSTR2 after its internalization when stimulated with SST-14 (Fig. 4AGo). Additionally, the transient nature of β-arrestin and SSTR2 on heterodimerization was supported by coimmunoprecipitation studies (Fig. 4Go, B–D). Stable complexes between SSTR2 and β-arrestin formed after extended treatment with L-779,976 in cells expressing SSTR2 alone; however, the interaction decreased significantly when SSTR5 was coexpressed. This effect was restricted to the SSTR2-specific agonist L-779,976 because treatment with SST-14 in either cell type did not alter the coimmunoprecipitation patterns of β-arrestin (Fig. 4Go, B–D).


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 4. Heterodimerization Alters the Association of β-Arrestin to SSTR2

HEK 293 cells were transiently transfected with 1 µg SSTR2, 1 µg SSTR5, 0.25 µg β-arrestin2-GFP, and 0.50 µg GRK2. Cells were treated 48 h after transfection with 10 nM of the indicated agonist. A, Confocal microscopy of cells treated with agonist showing β-arrestin (green) and SSTR2 (red) colocalization. Internalized receptor was imaged after cell permeabilization (P). Images represent at least three independent experiments. Scale bar, 10 µM. B, Coimmunoprecipitation of SSTR2 and β-arrestin from HEK 293 cells stably expressing the indicated receptor subtypes. Cells were treated with either SST-14 or L-779,976 for 5 or 20 min. Cell lysate was immunoprecipitated for SSTR2 and immunoblotted against β-arrestin. A band at approximately 50 kDa is represented as β-arrestin. C and D, Densitometric analysis on immunoblots presented in B. Data are represented as fold increase over control (0 min). Statistical analysis to determine significance from control was performed using two-way ANOVA and post hoc Bonferroni; ***, P < 0.001. Means ± SEM are representative of three independent experiments.

 
Interestingly, the heterodimer did not internalize as a complex, a fact that has been documented for other GPCR heterodimers (15). In fact, when cotransfected cells were treated with L-779,976, only SSTR2 was prompted to internalize (Fig. 5Go). Both SSTR2 and SSTR5 did internalize when stimulated with SST-14, agonist to both receptor subtypes (Fig. 5Go). Although heterodimerization may affect the sequestration of β-arrestin it did not affect the internalization of SSTR5, possibly implying the absence of heterologous events.


Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 5. Agonist-Promoted Internalization of SSTR2 and SSTR5

Confocal microscopy of HEK 293 cells stably expressing SSTR2 (red) and SSTR5 (green) after agonist stimulation. Cells were treated with 10 nM of the indicated agonist for 20 min, fixed, and then permeabilized (P) before immunocytochemistry. Nuclei were counterstained with DAPI (blue). Images are a representation of at least three independent experiments. Scale bar, 10 µm.

 
Trafficking of SSTR2 after Heterodimerization
GPCR recycling rates have been causally linked to changes in the association of β-arrestin (24). Stable associations between β-arrestin and the receptor result in slow recycling rates, contrary to transient interactions, where recycling times are shortened. Furthermore, a greater portion of receptor is sent to lysosomal compartments to be degraded, when β-arrestin is stably associated. Given the effects of heterodimerization on the association of β-arrestin to SSTR2, we sought to determine whether these changes were also reflected in receptor recycling rates. HEK 293 cells expressing SSTR2 or coexpressing SSTR2 and SSTR5 were treated with agonist and imaged by confocal microscopy to measure receptor trafficking (Fig. 6Go). When SSTR2-expressing cells were treated with either agonist SST-14 or L-779,976, internalization was evident in permeabilized cells after 20 or 40 min stimulation, accompanied by decreased cell surface expression levels (Fig. 6Go). A similar occurrence transpired when HEK 293 cells coexpressing SSTR2 and SSTR5 were treated with SST-14 for either 20 or 40 min (Fig. 6Go). Punctate staining representing internalized receptor was consistently found in perinuclear regions. Similarly, perinuclear staining was also portrayed in cells coexpressing SSTR2 and SSTR5 subsequent to SST-14 treatment. A markedly distinct pattern of SSTR2 localization ensued when coexpressing cells were treated with L-779,976 (Fig. 6Go). After 20 min activation with L-779,976, SSTR2 was found concentrated intracellularly; however, unlike SSTR2-expressing cells, extended stimulation (40 min) presented with an overall decrease in internalized receptor, with staining primarily localized around the cell periphery and not perinuclear (Fig. 6Go). Accordingly, coexpressing cells also demonstrated increases in cell surface expression after prolonged activation with L-779,976 (Fig. 6Go).


Figure 6
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 6. Heterodimerization Increases the Recycling of SSTR2

Confocal microscopic images of SSTR2 (red) trafficking after agonist stimulation. HEK 293 cells expressing SSTR2 or SSTR2 and SSTR5 were treated with 10 nM agonist SST-14 or L-779,976 for 20 or 40 min. Immunostaining of SSTR2 was performed using monoclonal anti-HA antibody followed by incubation with anti-mouse IgG antibody conjugated to fluorescein. Internalized receptor was identified by immunocytochemistry in permeabilized (P) cells and cell surface localization in nonpermeabilized (NP) cells. Nuclei were counterstained with DAPI (blue). Images are representative of at least three independent experiments. Scale bar, 10 µm.

 
Heterodimers Potentiate but Do Not Synergize G-Protein Signaling
There have been several instances where heterodimerization of GPCRs has led to changes in signaling distinct from that of the individual receptor monomers/homodimers (15). In fact, most of these changes have been documented within immediate effector responses such as changes in adenylyl cyclase activity. However, few reports have shown a physiological implication for GPCR heterodimerization (15, 26, 27, 28). To seek a functional relevance for the SSTR2/SSTR5 heterodimer, we began by studying the SSTR-induced inhibition of adenylyl cyclase (1, 2). We compared the cAMP efficiency curves of all three agonists from HEK 293 cells stably expressing one or both of the receptor subtypes. The SSTR2-specific agonist L-779,976 was approximately 20-fold more efficient at inhibiting adenylyl cyclase in cotransfectants than in cells expressing SSTR2 alone (Fig. 7AGo and Table 1Go). This increase in potency in cotransfected cells was exclusive to L-779,976 treatment, because the EC50 values for either the SSTR5-specific agonist L-817,818 or SST-14 were not significantly altered (Fig. 7Go, B and C, and Table 1Go). Heterodimerization did not result in a synergistic effect on adenylyl cyclase coupling, because the total inhibition of cAMP synthesis was unchanged (Fig. 7DGo). Congruently, GTP coupling as measured from the membranes of cotransfected HEK 293 cells did not significantly differ among the various agonist treatments (Fig. 7EGo). Binding of GTP was dependent on the coupling of G{alpha}i G proteins, because the effect was blocked by pretreatment with pertussis toxin (PTX). Although heterodimerization enhanced adenylyl cyclase coupling efficiency, the effect was not synergistic, because neither the total inhibition of cAMP synthesis nor the binding of GTP was affected.


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 7. Heterodimerization and Effector Coupling

Inhibition of forskolin-stimulated cAMP synthesis in HEK 293 cells stably expressing SSTR2 and SSTR5 individually or in combination and treated with increasing concentrations of L-779,976 (A), SST-14 (B), and L-817,818 (C) calculated as a percentage of maximal inhibition. Data were plotted and analyzed after a sigmoidal dose-response equation using Graph Pad Prism 4.0. D, Total inhibition of cAMP production after 10 nM treatment of each agonist in stably transfected cells. E, GTP binding of membranes extracted from HEK 293 cells stably expressing SSTR2 and SSTR5 treated with 10 nM of the indicated agonists. Binding was inhibited when membranes were pretreated with PTX. Statistical analysis was performed using ANOVA and post hoc Dunnett’s to compare for significance from PTX-treated membranes. Means ± SEM are representative of at least three independent experiments performed in duplicate. ***, P < 0.001.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Comparison of Adenylyl Cyclase Coupling Efficiencies by SSTR Agonists

 
Heterodimerization Enhances ERK1/2 Phosphorylation
Phosphorylation of ERK1/2 was shown as a prerequisite to the growth-inhibitory effects of SST, a property predominantly associated with SSTR2 activation (29, 30). To determine the effects of SSTR heterodimerization on ERK1/2 activity, we measured its phosphorylation in a time-dependent manner upon activation of SSTR2 in our single- and double-expressing cell lines. Treatment of HEK 293 cells stably expressing SSTR2 with L-779,976 resulted in a phosphorylation maximum at approximately 5 min, which receded to basal levels at the end of a 40-min stimulation period. However, the effect was significantly enhanced and prolonged, an event that persisted beyond 40 min when SSTR5 was stably present (Fig. 8Go, A and B). The phosphorylation of ERK1/2 was highly dependent on G{alpha}i G proteins, because pretreatment of cells with PTX abrogated the signal (Fig. 8AGo). Alterations in the phosphorylation profiles of ERK1/2 in cotransfected cells were not apparent when treated with SST-14, agonist to both receptor subtypes, suggesting coactivation is not required to enhance the response (Fig. 8CGo). Interestingly, selective activation of SSTR5 with L-817,818 did not affect ERK1/2 activation, supporting the importance of SSTR2 in this signaling pathway (supplemental Fig. S2).


Figure 8
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 8. The Effects of Heterodimerization on ERK1/2 Phosphorylation

A, HEK 293 cells stably expressing SSTR2 or SSTR2 and SSTR5 were treated with 10 nM L-779,976 for the indicated times. In case of PTX, cells were pretreated 18 h before agonist stimulation. Cell lysates were subjected to Western blotting and immunoblotted for ERK1/2 proteins. B, Densitometry was performed on phosphorylated ERK1/2 immunoblots in A. β-Tubulin was used to standardize for protein loading. Data were analyzed by ANOVA and post hoc Dunnett’s to compare for significance over basal; **, P < 0.01. Two-way ANOVA was performed to compare the significance of ERK1/2 phosphorylation between monotransfected and cotransfected cells; ##, P < 0.01; ###, P < 0.001. Means ± SEM are representative of three independent experiments. C, HEK 293 cells stably coexpressing SSTR2 and SSTR5 were treated with 10 nM SST-14 for the indicated times. Cell lysate was collected, subjected to Western blotting, and immunoblotted for the designated proteins.

 
Up-Regulation of p27Kip1 and Cell Growth Inhibition Are Increased on Heterodimerization
The growth-inhibitory effects of SST and its analogs have been characterized on both normal and tumor cells, a property shown to occur both in vitro and in vivo (3). The intracellular pathway elicited by activation of SSTR2 leading to G1 cell-cycle arrest, have involved MAPKs and up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 (29). Interference of this pathway by the MAPK kinase-1/2 inhibitor PD98059 abrogates p27Kip1 up-regulation and growth inhibition (30). To determine the effects of heterodimerization on SSTR2-evoked p27Kip1 expression, we compared the treatments of SST-14 and L-779,976 on HEK 293 cells stably expressing SSTR2 alone or in combination with SSTR5. Cells were serum deprived for 24 h before incubation with agonist in the presence of fetal bovine serum (FBS) to simulate growth conditions. The addition of FBS significantly decreased the expression of p27Kip1, a property associated with the growth-promoting effects of the serum (Fig. 9Go, A and B). Treating coexpressing cells with SST-14 produced a concentration-dependent increase in p27Kip1 expression, an effect that reached a maximum at 100 nM. A similar response was procured with as little as 1 nM L-779,976, a 100-fold increase in potency (Fig. 9AGo). This potency difference was not apparent when both agonists SST-14 and L-779,976 were used to treat HEK 293 cells stably expressing SSTR2 alone (Fig. 9BGo). As expected, selective stimulation of SSTR5 with L-817,818 in coexpressing cells did not affect p27Kip1 induction, given its inability to activate ERK1/2 (supplemental Fig. S3).


Figure 9
View larger version (44K):
[in this window]
[in a new window]

  		Fig. 9. Heterodimerization Potentiates the Up-Regulation of p27Kip1

A, Upper panel, HEK 293 cells stably expressing SSTR2 and SSTR5 were serum deprived for 24 h followed by treatment with the indicated concentrations of each agonist in the presence of 5% FBS, with FBS alone (FBS), or in its absence (control) for 3 h. Cell lysates were subjected to Western blot and probed for p27Kip1 expression. Lower panel, Densitometry on p27Kip1 immunoblots standardized for protein loading using β-tubulin. Data were analyzed by ANOVA and post hoc Dunnett’s to determine significance of FBS vs. treated; **, P < 0.01. B, Upper panel, immunoblot of lysates from HEK 293 cells stably expressing SSTR2 and treated with the indicated concentrations of each agonist and FBS for 3 h; lower panel, densitometry on p27Kip1 immunoblots. Statistical analysis was done using ANOVA and post hoc Dunnett’s to determine significance of FBS vs. treated; **, P < 0.01.

 
To determine whether increases in the up-regulation of p27Kip1 were associated with changes in cell physiology, we monitored the effects of heterodimerization on cell growth. Cell growth was diminished by approximately 50% after a 24-h incubation period with either agonist SST-14 or L-779,976 in cells stably expressing SSTR2 alone or in combination with SSTR5 (Fig. 10Go, A and B). The effect was not persistent, because continued treatment with SST-14 resulted in a consistent decline in growth inhibition over the course of a 72-h period, an indication that cell-cycle arrest was reversed. However, growth inhibition was sustained after treatment of L-779,976 on cells expressing both receptor subtypes, a property associated with the signaling events of SSTR heterodimers (Fig. 10BGo).


Figure 10
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 10. SSTR Heterodimers Enhance Cell Growth Inhibition

A and B, Growth inhibition of stably transfected HEK 293 cells treated with 1 nM SST-14 and L-779,976 for 24–72 h as a percentage of control in the presence of 5% FBS. Two-way ANOVA was used to determine the significance between drug treatments; *, P < 0.05. Means ± SEM are representative of at least four independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SST analogs are the mainstay in the medical treatment of acromegaly, because they are safe and well tolerated (4). Their function is highlighted by their effectiveness at normalizing GH and IGF-I levels, caused by hypersecreting pituitary adenomas. In addition, patients on SST analogs have shown significant reductions in tumor mass, and tumor shrinkage of greater than 50% can be seen when SST analogs are administered as stand-alone therapy, before any surgical or radiotherapy procedures (5, 6). These reports have broadened the potential use of SST analogs from hormone regulation to tumor control. In addition, GH-secreting pituitary adenomas do not undergo desensitization to treatment, because acromegalic patients rarely show any signs of tachyphylaxis despite years of SST analog therapy. This property is specific to tumors of the pituitary because neither islet cell nor carcinoid tumors share this feature; prolonged administration of SST analogs frequently results in desensitization and relapse, because symptoms invariably return (10, 31, 32).

The mechanisms underlying resistance to desensitization have not been fully elucidated; however, a functional association between SSTR2 and SSTR5, the two primary SSTRs expressed in these tumors (11, 12), has been proposed to account for this behavior (13). This is unlike islet cell tumors or carcinoids of the gut, where SSTR2 expression predominates (32). In the report by Sharif et al. (13), the internalization and trafficking of murine SSTR2 were modulated by the expression of SSTR5 in both ectopically and endogenously expressing cell systems. Moreover, the effect was dependent on the selective stimulation of SSTR2 and not both receptor subtypes. They observed reduced receptor internalization, increased receptor recycling, and a decrease in the desensitization of SSTR2 in coexpressing cells treated with the selective agonist L-779,976 compared with cells expressing SSTR2 alone or coexpressing cells treated with the pan-agonist SST-14. Additional support on the importance of both SSTR2 and SSTR5 expression on SST analog therapy comes from a report by Ballarè et al. (33) describing an acromegalic patient with a tumor harboring a mutant SSTR5 resistant to treatment despite the presence of SSTR2. These reports and others have suggested the formation of a putative SSTR2/SSTR5 heterodimer; however, a physical interaction has yet to be documented (34, 35).

Using both coimmunoprecipitation and pbFRET microscopy techniques, L-779,976 was determined to selectively regulate the SSTR2/SSTR5 heterodimer (Fig. 1Go). Neither concurrent stimulation nor selective activation of SSTR5 with SST-14 and L-817,818, respectively, stabilized this interaction. Given that SSTR2 exists as a homodimer that dissociates into monomers after activation with agonist (21, 36), it is possible that the uncoupling of the dimer could allow for alternative pairing with SSTR5. This could also explain the lower levels of heteromeric interaction seen under basal conditions and may suggest that SSTR2 has a higher affinity for interacting with itself when inactive. However, this mechanism fails to explain why activation by SST-14 does not regulate the heterodimer. One explanation for this discrepancy could be that although SST-14 binds both SSTR2 and SSTR5 relatively equally, activation of SSTR5 may favor the stabilization of homodimers. Indeed, when cells coexpressing SSTR2 and SSTR5 are treated with SST-14, SSTR5 dimers exhibit dose-dependent formation (Fig. 1DGo). Furthermore, this could also explain why treatment with the SSTR5-selective compound L-817,818 does not modulate heterodimerization. Selectivity in the heterodimerization of SSTRs has been reported earlier upon investigation of a SSTR4/SSTR5 heterodimer; coactivation by SST-14 in cells coexpressing both receptor subtypes did not induce an interaction (18). Although coactivation was not favorable for the heterodimerization of SSTR2 and SSTR5, it has been demonstrated as a requirement in the modulation of other GPCR combinations (19, 20, 28, 37, 38, 39, 40, 41, 42). For instance, in the heterodimerization of SSTR1 and SSTR5, concurrent activation was shown to foster an association (19, 20). Heterodimerization between the GH secretagogue and dopamine D1 receptors was shown to occur by coactivation using both coimmunoprecipitation and bioluminescence resonance energy transfer techniques (42). Furthermore, the interaction significantly increased dopamine-induced cAMP accumulation, a property that is suggested to play an important physiological role given that both receptors are coexpressed in neuronal subpopulations. In a recent report, heterodimerization of the CXCR4 chemokine receptor and the {delta}-opioid receptor in immune cells was stabilized by simultaneous addition of their ligands, whereas individual activation caused dissociation of the complex (28). Interestingly, the heterodimer suppresses signaling, suggesting a dominant negative effect on receptor function (28).

Activated GPCRs undergo desensitization followed by internalization to terminate signaling; this process usually requires phosphorylation of the receptor by a G protein-coupled receptor kinase, promoting high-affinity binding of β-arrestins (23). Once bound, β-arrestins prohibit the coupling of G protein to the receptor and promote internalization by recruiting several factors involved in this machinery (24). Only certain members of the SST receptor family exhibit high-affinity binding to β-arrestins (25). We demonstrate that activated SSTR2 and not SSTR5 are capable of initiating an interaction (Fig. 3AGo). This may indicate that although SSTR5 internalizes after activation with agonist, it does so without the involvement of β-arrestin. The mechanisms underlying the internalization of SSTR5 are still unclear; however, evidence suggests that it may be occurring through clathrin-coated pits (43, 44, 45). GPCRs that do interact with β-arrestins are further categorized as class A or class B, depending on their mode of interaction. Although both classes of GPCRs differ in their affinity for β-arrestin subtypes, they are most notably distinguished based on their avidity to β-arrestin; β-arrestin rapidly dissociates from class A receptors during internalization but remains associated with class B receptors, because it is found colocalized in the form of endosomes within cells (24). When HEK 293 cells expressing SSTR2 are stimulated with either agonist SST-14 or L-779,976 for an extended period, β-arrestin is prompted to aggregate and remain with the receptor throughout internalization. Treatment of cells coexpressing both receptor subtypes with SST-14 results in a similar feature; β-arrestin and SSTR2 colocalize in the form of endosomes. However, when treatment consisted of L-779,976 in lieu of SST-14, SSTR2 internalizes without β-arrestin associating. In fact, within minutes of treatment, β-arrestin translocates to SSTR2 at the cell surface but quickly dissociates because there is no colocalization after receptor internalization (Figs. 3BGo and 4Go). This is analogous to class A receptors where β-arrestin does not remain as a complex throughout endocytosis.

Changes in the association of β-arrestin to the receptor during internalization are known to directly affect recycling rates; class A receptors recycle to the plasma membrane much quicker than class B receptors and are less likely to be degraded (24). Indeed, heterodimerization does affect SSTR2 recycling rates. After a 20-min stimulus with L-779,976, SSTR2 internalizes in both single- and double-expressing cell lines (Fig. 6Go). However, the striking difference arises when coexpressing cells are treated with L-779,976 for extended periods. Under these conditions, an increase in the cell surface localization of SSTR2 is observed, with a concomitant decrease in intracellular receptor. These results are consistent with those of Sharif et al. (13), where extensive loss of cell surface SSTR2 is seen after stimulation of coexpressing with SST-14 and not with the SSTR2-specific agonist L-779,976, which causes SSTR2 to be recycled back to the cell surface. In a report by Terrillon et al. (46), a similar incidence was identified with the vasopressin V1a receptor, which upon heterodimerization with the vasopressin receptor subtype V2, reverted its pattern of β-arrestin recruitment and endocytic recycling from class A to class B. In case of the neurotensin receptors, heterodimerization between NTS1 and NTS2 influenced the subcellular distribution and capacity to down-regulate agonist-stimulated NTS1 (47). Similarly, heterodimerization between β-adrenergic receptor subtypes β1AR and β2AR and the SSTR1 and SSTR5 subtypes affect ligand-induced receptor internalization (18, 48).

Several lines of evidence implicate SSTRs in tumor regulation, in particular the subtype SSTR2, because it is well documented for its antiproliferative effects and is commonly expressed in many tumors (3). Inhibiting cell proliferation by SSTR2 activation involves the up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 (29), an important factor in pituitary tumorigenesis (49, 50, 51, 52, 53, 54). Moreover, a mechanism describing the up-regulation of p27Kip1 involved an ERK1/2-dependent pathway (30). Rapid recycling of SSTR2 could provide a mechanism for the observed increases in ERK1/2 phosphorylation (Fig. 8Go). This could also explain the increased potency of L-779,976 to up-regulate the cyclin-dependent kinase inhibitor p27Kip1 and prolong cell growth inhibition (Figs. 9Go and 10Go).

The SSTR2/SSTR5 heterodimer also increased the potency of L-779,976 to inhibit adenylyl cyclase activity by approximately 20-fold (Fig. 7Go and Table 1Go). Similar findings were reported for the SSTR1/SSTR5 heterodimer (20) and suggest that heterodimerization may also provide positive cooperativity to receptor function. Indeed, heterodimerization of several other receptor combinations including the dopamine D2 receptor and SSTR5 were also reported to enhance GPCR signaling (26, 55, 56, 57, 58, 59, 60). Although changes in β-arrestin association and endocytic recycling after heterodimerization could provide a mechanism for the observed increases in ERK activation and p27Kip1 up-regulation, positive cooperativity in G protein signaling may also contribute (Fig. 7Go and Table 1Go). A PTX-sensitive G{alpha}i G protein-mediated signaling complex has been described in orchestrating ERK1/2 activation and cell growth inhibition after activation of SSTR2 (30). Because the carboxyl-terminal tail of GPCRs is an important domain with which both G proteins and β-arrestin interact, it is possible that heterodimerization may alter the properties of this motif, allowing for changes in cooperativity or β-arrestin recruitment. Modifications could arise either by structural changes in the carboxyl-terminal tail itself or through its phosphorylation, because both are involved and presumed to affect the avidity of β-arrestin and G protein to the receptor (24). However, despite the possibility that each of the above mentioned mechanisms may contribute to the enhanced signaling observed after heterodimerization, further investigation is required to mechanistically link the pathways of relevance. Insight on the molecular determinants affected upon heterodimerization is required given the emerging roles of GPCR-interacting proteins on signal transduction (61).

It is noteworthy that although SSTR2 heterodimerizes with SSTR5, both protomers did not internalize as a complex, which suggests the process is restricted to only the activated protomer (Fig. 5Go). A similar phenomenon was reported for the SSTR2/SSTR3 heterodimer, where selective activation of SSTR2 resulted in its internalization without affecting the presence of SSTR3 (62). Furthermore, heteromeric complexes between dopamine D1 and adenosine A1 receptors were shown to disappear when treated with D1 receptor agonists by forming clusters that were devoid of A1 receptors (37). The µ- and {delta}-opioid receptor heterodimer was also shown not to endocytose as a complex but yet internalize as monomers when activated by agonists (63). Although formation of the prostaglandin-EP1 and β2-adrenergic heterodimer directly decreased β2-adrenergic receptor coupling to G{alpha}s, cross-regulation was not involved in attenuating β2-adrenergic receptor function because desensitization and internalization were factors affecting only the activated protomer (27). These results have important meaning because they imply that GPCR heterodimers are not necessarily static complexes in which both protomers are regulated as a unit.

Since the initial reports providing direct evidence for the heterodimerization of the GABAB receptor, a requirement for proper receptor trafficking and function (15), the pursuit of other receptor combinations has been sought. Although many such heterodimers have been reported, few have shown functional relevance. This makes their occurrence unclear, because there is no supporting evidence documenting an effect on cell physiology. In this study, we demonstrate that SSTR2 and SSTR5 form heterodimers, a process that leads to alterations in cell growth. Furthermore, agonist- and, more specifically, subtype-specific activation of SSTR2 modulates the interaction. Heterodimerization between SSTR2 and SSTR5 may provide a mechanism for the lack of tolerance seen with currently available SST analogs and their ability in controlling pituitary tumor growth. An understanding on the mechanics involved in GPCR dimerization could offer a rationale in future drug design.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials and Antibodies
The peptides SST-14 and [Leu8,D-Trp22,Tyr25] SST-28 were purchased from Bachem (Torrance, CA). The nonpeptide agonists L-779,976 and L-817,818 were provided by Dr. S. P. Rohrer from Merck & Co. (64). Fluorescein-conjugated mouse monoclonal antibody against HA (12CA5) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Rhodamine-conjugated and unconjugated anti-c-Myc monoclonal antibodies and mouse monoclonal anti-HA antibody were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Monoclonal anti-phospho-ERK1/2 and rabbit polyclonal ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA), and monoclonal anti-β-tubulin antibody from Sigma-Aldrich. Monoclonal anti-p27Kip1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A/G-agarose beads were purchased from Calbiochem, EMD Biosciences (Darmstadt, Germany). 4',6-Diamidino-2-phenylindole (DAPI) dihydrochloride was purchased from Molecular Probes, Inc. (Eugene, OR).

Constructs and Expressing Cell Lines
Stable transfections of HEK 293 cells expressing amino-terminally HA-tagged human SSTR2, c-Myc-tagged human SSTR5, or both receptors were prepared by Lipofectamine transfection reagent. Constructs expressing SSTR2 were made from the pCDNA3.1/Neo vector (neomycin resistance) and SSTR5 from the pCDNA3.1/Hygro vector (hygromycin resistance) as previously described (20, 21). Clones were selected and maintained in DMEM supplemented with 10% FBS and 700 µg/ml neomycin or 400 µg/ml hygromycin. Cotransfectants were maintained in medium containing both 700 µg/ml neomycin and 400 µg/ml hygromycin as previously described (20, 21). All cells were grown in a 37 C incubator with 5% CO2. Constructs for β-arrestin2-green fluorescent protein (GFP) and G protein-coupled receptor kinase 2 (GRK2) were kindly provided by Dr. Stéphane A. Laporte (McGill University).

Saturation Analysis
Cells were harvested and homogenized using a glass homogenizer, and membranes were prepared by centrifugation as previously described (20, 21). Saturation binding studies were performed with 20–40 µg membrane protein collected from HEK 293 cells stably expressing the receptor constructs and 125I-labeled [Leu8,D-Trp22,Tyr25] SST-28 radioligand (50–2000 pM) in 50 mM HEPES (pH 7.5), 2 mM CaCl2, 5 mM MgCl2, 0.5% BSA, 0.02% phenylmethylsulfonyl fluoride, and 0.02% bacitracin (binding buffer) for 30 min at 37 C. Incubations were terminated by the addition of ice-cold binding buffer. Membrane pellets were quantified for radioactivity using an LKB {gamma}-counter (LKB-Wallach, Turku, Finland). Binding data were analyzed with Prism 4.0 (GraphPad Software, San Diego, CA) by nonlinear regression analysis.

Coupling to Adenylyl Cyclase and GTP-Binding Assay
Stably transfected HEK 293 cells were grown in six-well plates and tested for receptor coupling to adenylyl cyclase by incubation for 30 min with 20 µM forskolin and 0.5 mM 3-isobutyl-1-methylxanthine with or without agonists (10–11 to 10–7 M) at 37 C as previously described (20). Cells were then scraped in 0.1 N HCl and quantified for cAMP by RIA using a cAMP kit (Inter Medico, Markham, Ontario, Canada) following the manufacturer’s guidelines. Samples were measured for radioactivity using an LKB β-scintillation counter (LKB-Wallach). Data were analyzed by nonlinear regression analysis using Prism 4.0 (Graph Pad Software). SEMS are representative of at least three independent experiments done in duplicate.

The GTP-binding assay was performed by measuring the amount of [35S]GTP{gamma}S (GE Healthcare, Waukesha, WI) bound to membranes from HEK 293 cells stably coexpressing SSTR2 and SSTR5. In 100 µl GTP assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, and 10 µM GDP), 50 µg membrane protein and 10 pM [35S]GTP{gamma}S were added in culture tubes with or without 10 nM agonist (SST-14, L-779,976, and L-817,818). The reaction was incubated in a 30 C water bath shaking for 1 h. To inactivate G{alpha}i G proteins, membrane was pretreated with 0.5 µg activated PTX [10 mM dithiothreitol, 10 µM ATP, PBS (pH 7.4) for 30 min at 30 C] for 1 h at 30 C before GTP binding. The reaction was terminated by the addition of 1 ml ice-cold GTP assay buffer. After centrifugation, membrane pellets were washed thrice in assay buffer before the addition of 7 ml scintillation fluid and counted using an LKB β-scintillation counter (LKB-Wallach).

PbFRET Microscopy and Immunocytochemistry
The pbFRET experiments were performed on HEK 293 cells as previously described (20, 21, 22). The effective FRET efficiency (E) was calculated in terms of a percentage based upon the photobleaching (pb) time constants of the donor (D) taken in the absence (D – A) and presence (D + A) of acceptor (A) according to E = 1 – ({tau}D-A/{tau}D+A) x 100. HEK 293 cells were seeded on glass coverslips for 24 h, treated with 10 nM of agonist for 10 min at 37 C, and fixed with 4% paraformaldehyde for 20 min on ice and processed for immunocytochemistry. Antibodies used were mouse monoclonal anti-HA conjugated to fluorescein directed to SSTR2 as the donor and monoclonal anti-c-Myc conjugated to rhodamine directed to SSTR5 as the acceptor. The area consisting of the plasma membrane was used to analyze the photobleaching decay rate on a pixel-by-pixel basis as described earlier.

Coimmunoprecipitation and Western Blot
Membrane protein (500 µg) from stably transfected HEK 293 cells was treated with 10 nM agonist (SST-14, L-779,976, and L-817,818) in binding buffer (50 mM HEPES, 2 mM CaCl2, 5 mM MgCl2, pH 7.5) for 30 min at 37 C. After treatment, membrane protein was solubilized in 1 ml radioimmunoprecipitation assay buffer [150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, pH 8.0] for 1 h at 4 C. Samples were incubated with anti-HA antibody for immunoprecipitation and purified with protein A/G-agarose beads. Purified proteins were fractionated by electrophoresis on a 7% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (GE Healthcare) as previously described (20, 21). Immunoblotting for the heterodimer was performed using anti-c-Myc antibody (1:2000). Blocking of membrane, incubation of primary antibodies, incubation of secondary antibodies, and detection by chemiluminescence were performed with the ECL Western blotting detection kit (Amersham) according to the manufacturer’s instructions. Images were captured using an Alpha Innotech FluorChem 8800 (Alpha Innotech Co., San Leandro, CA) gel box imager, and densitometry was carried out using FluorChem software (Alpha Innotech).

For MAPK signaling, HEK 293 cells stably coexpressing SSTR2 and SSTR5 were treated with 10 nM of each agonist for the indicated times. The reaction was terminated using ice-cold Dulbecco’s PBS followed by solubilization in radioimmunoprecipitation assay buffer. Lysate protein was separated on an 8% polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Immunoblotting for phosphorylated ERK1/2 was performed using phosphospecific antibodies. Phosphorylation levels were quantified by densitometry using FluorChem software and normalized for protein loading using β-tubulin. The same procedure was repeated for the expression of p27Kip1; however, immunoblotting was performed using monoclonal anti-p27Kip1 antibodies. To standardize for protein loading, all membranes were reprobed for β-tubulin using Reblot Plus (Chemicon International, Temecula, CA).

Assay of β-Arrestin Translocation
HEK 293 cells were seeded in six-well plates with coverslips for 24 h before transient transfection with SSTR2, SSTR5, or both receptor subtypes along with β-arrestin2-GFP (β-arrestin) and GRK2 cDNAs using Lipofectamine transfection reagent. Cells were treated with 10 nM agonist 48 h after transfection for the indicated times. SSTR2 was localized using an anti-HA antibody and antimouse IgG conjugated to cy3 (red), whereas β-arrestin (green) was identified by direct excitation of the GFP molecule using 488-nm laser light. Cells were fixed in 4% paraformaldehyde before being subjected to immunocytochemistry and imaged using a Zeiss 510 confocal microscope. Cells treated with SST-14 and L-779,976 for 20 min were permeabilized using 0.2% Triton X-100 for 10 min before immunocytochemistry.

Cell Growth Inhibition
HEK 293 cells stably expressing SSTR2 and SSTR5 were seeded at a density of 5000 cells per well in 96-well plates for 24 h. Cells were then serum deprived for 24 h before treatment with 1 nM of either agonist SST-14 or L-779,976 in the presence of 5% FBS for the indicated time periods. As a control, cells were given FBS in the absence of drug to compare for cell growth. Cell viability was measured using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay protocol. Briefly, 10 µl 5 mg/ml MTT (thiazolyl blue tetrazolium bromide; Sigma-Aldrich) solution was added and left to incubate for 2 h at 37 C and 5% CO2. When the formazan precipitate was formed, it was dissolved in 100 µl detergent solution (N,N-dimethylformamide, 20% SDS solution). The absorbance was measured in a microplate spectrophotometer at 550 nm.

Statistical Analysis
Data were analyzed with the indicated tests using GraphPad Prism 4.0. Statistical differences were taken at P values < 0.05.


   ACKNOWLEDGMENTS
 
We thank S. A. Laporte for the β-arrestin-GFP and GRK2 constructs, A. Abdallah for his technical assistance, and G. H. Hendy for his support and critical feedback on the manuscript.


   FOOTNOTES
 
This work was supported by the Canadian Institute for Health Research (Grants MOP74465 and MOP6196). M.G. holds a studentship from the Fonds de la Recherche en Santé du Québec.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; FBS, fetal bovine serum; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GRK2, G protein-coupled receptor kinase 2; HA, hemagglutinin; pbFRET, photobleaching fluorescence resonance energy transfer; PTX, pertussis toxin; SDS, sodium dodecyl sulfate; SST, somatostatin; SSTR, SST receptor.

Received for publication July 2, 2007. Accepted for publication July 15, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Patel YC 1999 Somatostatin and its receptor family. Front Neuroendocrinol 20:157–198[CrossRef][Medline]
  2. Moller LN, Stidsen CE, Hartmann B, Holst JJ 2003 Somatostatin receptors. Biochim Biophys Acta 1616:1–84[Medline]
  3. Lamberts SW, de Herder WW, Hofland LJ 2002 Somatostatin analogs in the diagnosis and treatment of cancer. Trends Endocrinol Metab 13:451–457[CrossRef][Medline]
  4. Tichomirowa MA, Daly AF, Beckers A 2005 Treatment of pituitary tumors: somatostatin. Endocrine 28:93–100[CrossRef][Medline]
  5. Bevan JS 2005 The antitumoral effects of somatostatin analog therapy in acromegaly. J Clin Endocrinol Metab 90:1856–1863[Abstract/Free Full Text]
  6. Melmed S, Sternberg R, Cook D, Klibanski A, Chanson P, Bonert V, Vance ML, Rhew D, Kleinberg D, Barkan A 2005 A critical analysis of pituitary tumor shrinkage during primary medical therapy in acromegaly. J Clin Endocrinol Metab 90:4405–4410[Abstract/Free Full Text]
  7. Ferrante E, Pellegrini C, Bondioni S, Peverelli E, Locatelli M, Gelmini P, Luciani P, Peri A, Mantovani G, Bosari S, Beck-Peccoz P, Spada A, Lania A 2006 Octreotide promotes apoptosis in human somatotroph tumor cells by activating somatostatin receptor type 2. Endocr Relat Cancer 13:955–962[Abstract/Free Full Text]
  8. Zatelli MC, Piccin D, Ambrosio MR, Bondanelli M, degli Uberti EC 2006 Antiproliferative effects of somatostatin analogs in pituitary adenomas. Pituitary 9:27–34[CrossRef][Medline]
  9. Resmini E, Dadati P, Ravetti JL, Zona G, Spaziante R, Saveanu A, Jaquet P, Culler MD, Bianchi F, Rebora A, Minuto F, Ferone D 2007 Rapid pituitary tumor shrinkage with dissociation between antiproliferative and antisecretory effects of a long-acting octreotide in an acromegalic patient. J Clin Endocrinol Metab 92:1592–1599[Abstract/Free Full Text]
  10. Hofland LJ, Lamberts SW 2003 The pathophysiological consequences of somatostatin receptor internalization and resistance. Endocr Rev 24:28–47[Abstract/Free Full Text]
  11. Jaquet P, Saveanu A, Gunz G, Fina F, Zamora AJ, Grino M, Culler MD, Moreau JP, Enjalbert A, Ouafik LH 2000 Human somatostatin receptor subtypes in acromegaly: distinct patterns of messenger ribonucleic acid expression and hormone suppression identify different tumoral phenotypes. J Clin Endocrinol Metab 85:781–792[Abstract/Free Full Text]
  12. Park C, Yang I, Woo J, Kim S, Kim J, Kim Y, Sohn S, Kim E, Lee M, Park H, Jung J, Park S 2004 Somatostatin (SRIF) receptor subtype 2 and 5 gene expression in growth hormone-secreting pituitary adenomas: the relationship with endogenous SRIF activity and response to octreotide. Endocr J 51:227–236[CrossRef][Medline]
  13. Sharif N, Gendron L, Wowchuk J, Sarret P, Mazella J, Beaudet A, Stroh T 2007 Coexpression of somatostatin receptor subtype 5 affects internalization and trafficking of somatostatin receptor subtype 2. Endocrinology 148:2095–2105[Abstract/Free Full Text]
  14. Terrillon S, Bouvier M 2004 Roles of G-protein-coupled receptor dimerization. EMBO Rep 5:30–34[CrossRef][Medline]
  15. Prinster SC, Hague C, Hall RA 2005 Heterodimerization of G protein-coupled receptors: specificity and functional significance. Pharmacol Rev 57:289–298[Abstract/Free Full Text]
  16. AbdAlla S, Lother H, el Massiery A, Quitterer U 2001 Increased AT1 receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nat Med 7:1003–1009[CrossRef][Medline]
  17. AbdAlla S, Lother H, Langer A, el Faramawy Y, Quitterer U 2004 Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis. Cell 119:343–354[CrossRef][Medline]
  18. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC 2000 Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem 275:7862–7869[Abstract/Free Full Text]
  19. Patel RC, Kumar U, Lamb DC, Eid JS, Rocheville M, Grant M, Rani A, Hazlett T, Patel SC, Gratton E, Patel YC 2002 Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells. Proc Natl Acad Sci USA 99:3294–3299[Abstract/Free Full Text]
  20. Grant M, Patel RC, Kumar U 2004 The role of subtype-specific ligand binding and the C-tail domain in dimer formation of human somatostatin receptors. J Biol Chem 279:38636–38643[Abstract/Free Full Text]
  21. Grant M, Collier B, Kumar U 2004 Agonist-dependent dissociation of human somatostatin receptor 2 dimers: a role in receptor trafficking. J Biol Chem 279:36179–36183[Abstract/Free Full Text]
  22. Patel RC, Lange DC, Patel YC 2002 Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes. Methods 27:340–348[CrossRef][Medline]
  23. Tsao P, Cao T, von Zastrow M 2001 Role of endocytosis in mediating downregulation of G-protein-coupled receptors. Trends Pharmacol Sci 22:91–96[Medline]
  24. Lefkowitz RJ, Shenoy SK 2005 Transduction of receptor signals by β-arrestins. Science 308:512–517[Abstract/Free Full Text]
  25. Tulipano G, Stumm R, Pfeiffer M, Kreienkamp HJ, Hollt V, Schulz S 2004 Differential β-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes. J Biol Chem 279:21374–21382[Abstract/Free Full Text]
  26. Waldhoer M, Fong J, Jones RM, Lunzer MM, Sharma SK, Kostenis E, Portoghese PS, Whistler JL 2005 A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc Natl Acad Sci USA 102:9050–9055[Abstract/Free Full Text]
  27. McGraw DW, Mihlbachler KA, Schwarb MR, Rahman FF, Small KM, Almoosa KF, Liggett SB 2006 Airway smooth muscle prostaglandin-EP1 receptors directly modulate β2-adrenergic receptors within a unique heterodimeric complex. J Clin Invest 116:1400–1409[CrossRef][Medline]
  28. Pello OM, Martinez-Munoz L, Parrillas V, Serrano A, Rodriguez-Frade JM, Toro MJ, Lucas P, Monterrubio M, Martinez AC, Mellado M 2008 Ligand stabilization of CXCR4/{delta}-opioid receptor heterodimers reveals a mechanism for immune response regulation. Eur J Immunol 38:537–549[CrossRef][Medline]
  29. Pages P, Benali N, Saint-Laurent N, Esteve JP, Schally AV, Tkaczuk J, Vaysse N, Susini C, Buscail L 1999 sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of SHP-1. J Biol Chem 274:15186–15193[Abstract/Free Full Text]
  30. Lahlou H, Saint-Laurent N, Esteve JP, Eychene A, Pradayrol L, Pyronnet S, Susini C 2003 sst2 somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation. J Biol Chem 278:39356–39371[Abstract/Free Full Text]
  31. Lamberts SW, van der Lely AJ, de Herder WW, Hofland LJ 1996 Octreotide. N Engl J Med 334:246–254[Free Full Text]
  32. de Herder WW, Hofland LJ, van der Lely AJ, Lamberts SW 2003 Somatostatin receptors in gastroentero-pancreatic neuroendocrine tumours. Endocr Relat Cancer 10:451–458[Abstract]
  33. Ballare E, Persani L, Lania AG, Filopanti M, Giammona E, Corbetta S, Mantovani S, Arosio M, Beck-Peccoz P, Faglia G, Spada A 2001 Mutation of somatostatin receptor type 5 in an acromegalic patient resistant to somatostatin analog treatment. J Clin Endocrinol Metab 86:3809–3814[Abstract/Free Full Text]
  34. Ren SG, Taylor J, Dong J, Yu R, Culler MD, Melmed S 2003 Functional association of somatostatin receptor subtypes 2 and 5 in inhibiting human growth hormone secretion. J Clin Endocrinol Metab 88:4239–4245[Abstract/Free Full Text]
  35. Ben-Shlomo A, Wawrowsky KA, Proekt I, Wolkenfeld NM, Ren SG, Taylor J, Culler MD, Melmed S 2005 Somatostatin receptor type 5 modulates somatostatin receptor type 2 regulation of adrenocorticotropin secretion. J Biol Chem 280:24011–24021[Abstract/Free Full Text]
  36. Duran-Prado M, Bucharles C, Gonzalez BJ, Vazquez-Martinez R, Martinez-Fuentes AJ, Garcia-Navarro S, Rhodes SJ, Vaudry H, Malagon MM, Castano JP 2007 Porcine somatostatin receptor 2 displays typical pharmacological sst2 features but unique dynamics of homodimerization and internalization. Endocrinology 148:411–421[Abstract/Free Full Text]
  37. Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela EI, Rondin S, Lew JY, Watson S, Zoli M, Agnati LF, Verniera P, Lluis C, Ferre S, Fuxe K, Franco R 2000 Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci USA 97:8606–8611[Abstract/Free Full Text]
  38. Mellado M, Rodriguez-Frade JM, Vila-Coro AJ, Fernandez S, Martin de Ana A, Jones DR, Toran JL, Martinez AC 2001 Chemokine receptor homo- or heterodimerization activates distinct signaling pathways. EMBO J 20:2497–2507[CrossRef][Medline]
  39. Yoshioka K, Saitoh O, Nakata H 2002 Agonist-promoted heteromeric oligomerization between adenosine A1 and P2Y1 receptors in living cells. FEBS Lett 523:147–151[CrossRef][Medline]
  40. Rodriguez-Frade JM, del Real G, Serrano A, Hernanz-Falcon P, Soriano SF, Vila-Coro AJ, de Ana AM, Lucas P, Prieto I, Martinez AC, Mellado M 2004 Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor. EMBO J 23:66–76[CrossRef][Medline]
  41. Kearn CS, Blake-Palmer K, Daniel E, Mackie K, Glass M 2005 Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors enhances heterodimer formation: a mechanism for receptor cross-talk? Mol Pharmacol 67:1697–1704[Abstract/Free Full Text]
  42. Jiang H, Betancourt L, Smith RG 2006 Ghrelin amplifies dopamine signaling by cross talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype 1 heterodimers. Mol Endocrinol 20:1772–1785[Abstract/Free Full Text]
  43. Hukovic N, Panetta R, Kumar U, Rocheville M, Patel YC 1998 The cytoplasmic tail of the human somatostatin receptor type 5 is crucial for interaction with adenylyl cyclase and in mediating desensitization and internalization. J Biol Chem 273:21416–21422[Abstract/Free Full Text]
  44. Hukovic N, Rocheville M, Kumar U, Sasi R, Khare S, Patel YC 1999 Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail. J Biol Chem 274:24550–24558[Abstract/Free Full Text]
  45. Stroh T, Jackson AC, Sarret P, Dal Farra C, Vincent JP, Kreienkamp HJ, Mazella J, Beaudet A 2000 Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand- induced endocytosis. Endocrinology 141:354–365[Abstract/Free Full Text]
  46. Terrillon S, Barberis C, Bouvier M 2004 Heterodimerization of V1a and V2 vasopressin receptors determines the interaction with β-arrestin and their trafficking patterns. Proc Natl Acad Sci USA 101:1548–1553[Abstract/Free Full Text]
  47. Perron A, Sharif N, Sarret P, Stroh T, Beaudet A 2007 NTS2 modulates the intracellular distribution and trafficking of NTS1 via heterodimerization. Biochem Biophys Res Commun 353:582–590[CrossRef][Medline]
  48. Lavoie C, Mercier JF, Salahpour A, Umapathy D, Breit A, Villeneuve LR, Zhu WZ, Xiao RP, Lakatta EG, Bouvier M, Hebert TE 2002 β1/β2-Adrenergic receptor heterodimerization regulates β2-adrenergic receptor internalization and ERK signaling efficacy. J Biol Chem 277:35402–35410[Abstract/Free Full Text]
  49. Qian X, Jin L, Grande JP, Lloyd RV 1996 Transforming growth factor-β and p27 expression in pituitary cells. Endocrinology 137:3051–3060[Abstract]
  50. Qian X, Jin L, Kulig E, Lloyd RV 1998 DNA methylation regulates p27kip1 expression in rodent pituitary cell lines. Am J Pathol 153:1475–1482[Abstract/Free Full Text]
  51. Takeuchi S, Koeffler HP, Hinton DR, Miyoshi I, Melmed S, Shimon I 1998 Mutation and expression analysis of the cyclin-dependent kinase inhibitor gene p27/Kip1 in pituitary tumors. J Endocrinol 157:337–341[Abstract]
  52. Lidhar K, Korbonits M, Jordan S, Khalimova Z, Kaltsas G, Lu X, Clayton RN, Jenkins PJ, Monson JP, Besser GM, Lowe DG, Grossman AB 1999 Low expression of the cell cycle inhibitor p27Kip1 in normal corticotroph cells, corticotroph tumors, and malignant pituitary tumors. J Clin Endocrinol Metab 84:3823–3830[Abstract/Free Full Text]
  53. Bamberger CM, Fehn M, Bamberger AM, Ludecke DK, Beil FU, Saeger W, Schulte HM 1999 Reduced expression levels of the cell-cycle inhibitor p27Kip1 in human pituitary adenomas. Eur J Endocrinol 140:250–255[Abstract]
  54. Korbonits M, Chahal HS, Kaltsas G, Jordan S, Urmanova Y, Khalimova Z, Harris PE, Farrell WE, Claret FX, Grossman AB 2002 Expression of phosphorylated p27(Kip1) protein and Jun activation domain-binding protein 1 in human pituitary tumors. J Clin Endocrinol Metab 87:2635–2643[Abstract/Free Full Text]
  55. Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Patel YC 2000 Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288:154–157[Abstract/Free Full Text]
  56. AbdAlla S, Lother H, Quitterer U 2000 AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature 407:94–98[CrossRef][Medline]
  57. Scarselli M, Novi F, Schallmach E, Lin R, Baragli A, Colzi A, Griffon N, Corsini GU, Sokoloff P, Levenson R, Vogel Z, Maggio R 2001 D2/D3 dopamine receptor heterodimers exhibit unique functional properties. J Biol Chem 276:30308–30314[Abstract/Free Full Text]
  58. Yoshioka K, Saitoh O, Nakata H 2001 Heteromeric association creates a P2Y-like adenosine receptor. Proc Natl Acad Sci USA 98:7617–7622[Abstract/Free Full Text]
  59. Gomes I, Gupta A, Filipovska J, Szeto HH, Pintar JE, Devi LA 2004 A role for heterodimerization of µ and {delta} opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci USA 101:5135–5139[Abstract/Free Full Text]
  60. Zhu WZ, Chakir K, Zhang S, Yang D, Lavoie C, Bouvier M, Hebert TE, Lakatta EG, Cheng H, Xiao RP 2005 Heterodimerization of β1- and β2-adrenergic receptor subtypes optimizes β-adrenergic modulation of cardiac contractility. Circ Res 97:244–251[Abstract/Free Full Text]
  61. Bockaert J, Fagni L, Dumuis A, Marin P 2004 GPCR interacting proteins (GIP). Pharmacol Ther 103:203–221[CrossRef][Medline]
  62. Pfeiffer M, Koch T, Schroder H, Klutzny M, Kirscht S, Kreienkamp HJ, Hollt V, Schulz S 2001 Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst3 receptor function by heterodimerization with sst2A. J Biol Chem 276:14027–14036[Abstract/Free Full Text]
  63. Law PY, Erickson-Herbrandson LJ, Zha QQ, Solberg J, Chu J, Sarre A, Loh HH 2005 Heterodimerization of µ- and {delta}-opioid receptors occurs at the cell surface only and requires receptor-G protein interactions. J Biol Chem 280:11152–11164[Abstract/Free Full Text]
  64. Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor F, Mitra SW, Degrado SJ, Shu M, Klopp JM, Cai SJ, Blake A, Chan WW, Pasternak A, Yang L, Patchett AA, Smith RG, Chapman KT, Schaeffer JM 1998 Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry. Science 282:737–740[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
F. Barbieri, A. Pattarozzi, M. Gatti, C. Aiello, A. Quintero, G. Lunardi, A. Bajetto, A. Ferrari, M. D. Culler, and T. Florio
Differential efficacy of SSTR1, -2, and -5 agonists in the inhibition of C6 glioma growth in nude mice
Am J Physiol Endocrinol Metab, November 1, 2009; 297(5): E1078 - E1088.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. M. Luque, B. S. Soares, X.-d. Peng, S. Krishnan, J. Cordoba-Chacon, L. A. Frohman, and R. D. Kineman
Use of the Metallothionein Promoter-Human Growth Hormone-Releasing Hormone (GHRH) Mouse to Identify Regulatory Pathways that Suppress Pituitary Somatotrope Hyperplasia and Adenoma Formation due to GHRH-Receptor Hyperactivation
Endocrinology, July 1, 2009; 150(7): 3177 - 3185.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
D. Ferone, F. Gatto, M. Arvigo, E. Resmini, M. Boschetti, C. Teti, D. Esposito, and F. Minuto
The clinical-molecular interface of somatostatin, dopamine and their receptors in pituitary pathophysiology
J. Mol. Endocrinol., May 1, 2009; 42(5): 361 - 370.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grant, M.
Right arrow Articles by Kumar, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grant, M.
Right arrow Articles by Kumar, U.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals