QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.105.014985v1 69/1/45    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hague, C. Articles by Minneman, K. P. Search for Related Content PubMed PubMed Citation Articles by Hague, C. Articles by Minneman, K. P.

Heterodimers of _1B- and _1D-Adrenergic Receptors Form a Single Functional Entity

Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia

Received May 20, 2005; accepted September 29, 2005

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Heterologous expression of _1D-adrenergic receptors (_1D-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with _1B-ARs promotes surface localization of _1D-ARs. Here, we report that the _1B-/_1D-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of _1B-ARs and epitope-tagged _1D-ARs in both human embryonic kidney 293 and DDT₁MF-2 cells, _1D-AR binding sites were not detectable with the _1D-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect _1D-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the _1B-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in _1B/_1D-AR-expressing cells, revealing that _1D-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in _1B/_1D-AR-coexpressing cells. Stable coexpression of an uncoupled mutant _1B-AR (12) with _1D-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that _1B/_1D-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why _1D-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with _1B-ARs.

Numerous studies have now shown that GPCR heterodimerization is essential for proper expression and function of GABA_B (Marshall et al., 1999), taste (Nelson et al., 2001), olfactory (Hague et al., 2004b), and _1D-adrenergic receptors (ARs) (Hague et al., 2004c). The most convincing and thoroughly studied example to date of GPCR heterodimerization involves the formation of functional GABA_B receptors. It is now clear that GABA_BR1 and GABA_BR2 must heterodimerize to ensure trafficking of GABA_B receptors to the cell surface (Kaupmann et al., 1998; Marshall et al., 1999) at least partially through the masking of an endoplasmic reticulum (ER) retention signal located in the carboxyl-terminal tail of GABA_BR1 receptors (Margeta-Mitrovic et al., 2000). In addition, the formation of sweet taste receptors requires heterodimerization of T1R2 and T1R3 receptors (Nelson et al., 2001), and the M71 mouse olfactory receptor can achieve surface expression and become functional when heterodimerized with the ₂-AR (Hague et al., 2004b). In previous studies, we showed that _1D-AR heterodimerization with _1B-ARs was required to promote surface expression of the intracellularly retained _1D-AR (Hague et al., 2004c). These examples provide compelling evidence for GPCR heterodimerization in regulating GPCR cellular localization. However, with a handful of exceptions, such as - and -opioid (Jordan and Devi, 1999), D2 and D3 dopamine (Maggio et al., 2003), and _2A-/₁-adrenergic receptors (Xu et al., 2003), few examples of receptor heterodimerization causing significant pharmacological changes have been reported to date.

One longstanding mystery in the ₁-AR field has been the inability to detect _1D-AR binding sites in intact tissues with the _1D-AR-selective antagonist BMY 7378 (Yang et al., 1997, 1998), despite the fact that _1D-AR mRNA is as widely expressed throughout the body as mRNA for the _1A-AR and _1B-AR subtypes (Rokosh et al., 1994; Alonso-Llamazares et al., 1995; Scofield et al., 1995). Previous studies have suggested that _1D-AR mRNA may only be translated in response to specific stimuli, such as a loss of other ₁-AR subtypes (Turnbull et al., 2003) or hypertension (Ibarra et al., 2000). On the other hand, _1D-AR mRNA may be widely translated, but _1D-AR ligand binding may be masked or may exhibit altered properties in certain tissues. It has long been apparent that the _1D-AR is the most poorly coupled of all ₁-ARs (Theroux et al., 1996) and that one possible reason could be that it functions poorly without a binding partner, such as the _1B-AR. We report in this study that _1D-ARs coexpressed with _1B-ARs are undetectable with BMY 7378. Using immunochemical, biochemical, and pharmacological approaches, we found that _1D-/_1B-AR heterodimers act as a single entity with novel pharmacological properties, and each receptor subunit contributes a specific functional component to the complex.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Materials were obtained from the following sources: cDNAs for the wild-type human _1A-AR (Hirasawa et al., 1993) and human _1D-AR C-terminally tagged GFP constructs in pEGFP-N3 (Xu et al., 1999) were generously provided by Dr. Gozoh Tsujimoto (National Children's Hospital, Tokyo, Japan), human _1B-AR cDNA (Ramarao et al., 1992) was a gift from Dr. Dianne Perez (Cleveland Clinic, Cleveland, OH), and human _1D-AR cDNA was cloned in our laboratory (Esbenshade et al., 1995); FLAG/GFP-tagged human _1D-ARs and ^1–79_1D-ARs were created previously in our laboratory (Vicentic et al., 2002; Hague et al., 2004a). Hamster 12_1B-AR in pCMV was a gift from Dr. Myron Toews (University of Nebraska Medical Center, Omaha, NE); -T1A and F18A mutants were a gift from Dr. Richard Lewis (Xenome Ltd., Queensland, Australia); HEK293 and DDT₁MF-2 cells were from American Type Culture Collection (Manassas, VA); 5-methylurapidil, niguldipine, BMY 7378, (–)-norepinephrine bitartrate, Dowex 1 Resin, horseradish peroxidase-conjugated anti-Flag M2 antibody, and bovine serum albumin were from Sigma-Aldrich (St. Louis, MO); [myo-³H]inositol was from American Radiolabeled Chemicals (St. Louis, MO); Lipofectamine 2000 transfection reagent, fetal bovine serum, and penicillin/streptomycin were from Invitrogen (Carlsbad, CA); enzyme-linked immunosorbent assay enhanced chemiluminescence was from Pierce Chemical (Rockford, IL); Vectashield mounting medium was from Vector Laboratories (Burlingame, CA); and Dulbecco's modified Eagle's medium was from Cellgro-Mediatech (Herndon, VA).

Cell Culture and Transfection. HEK293 and DDT₁MF-2 cells were propagated in Dulbecco's modified Eagle's medium with sodium pyruvate supplemented with 10% heat inactivated fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO₂. Confluent plates were subcultured at a ratio of 1:5 for transfection. HEK293 and DDT₁MF-2 cells were transfected with 10 µg of DNA of each construct for 3 h using Lipofectamine 2000 transfection reagent, and cells were used for experimentation 48 to 72 h after transfection. Stable transfection of receptors was obtained by selection with 400 µg/ml G418 (pcDNA3.1, pDT, and pEGFP vectors) or 200 µg/ml hygromycin (pREP4 vector).

Luminometer-Based Surface-Expression Assay. DDT₁MF-2 cells were split into poly(D-lysine)-coated 35-mm dishes and incubated with horseradish peroxidase-conjugated M2-anti-FLAG antibody in blocking buffer, and cell-surface luminescence was determined using a method described previously (Hague et al., 2004c).

Laser Confocal Microscopy. Cells were grown on sterile coverslips, fixed for 30 min with 2% paraformaldehyde in 0.1 M phosphate buffer, washed, mounted, and scanned with a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss GmbH, Heidelberg, Germany) as described previously (Hague et al., 2004c). For detecting GFP, fluorescein isothiocyanate fluorescence was excited using an argon laser at a wavelength of 488 nm, and the absorbed wavelength was detected for 510 to 520 nm for GFP.

Immunoprecipitation/Immunoblotting. DDT₁MF-2 cells expressing FLAG-_1D GFP ARs were harvested by scraping in ice-cold phosphate-buffered saline (PBS) and washed by repeated centrifugation and homogenization. Cell lysates were solubilized, immunoprecipitated with anti-FLAG M2 affinity resin, and probed using anti-FLAG M2 monoclonal antibodies as described previously (Uberti et al., 2003).

Radioligand Binding. Confluent 150-mm plates were washed with PBS (20 mM NaPO₄ and 154 mM NaCl, pH 7.6) and harvested by scraping. Cells were collected by centrifugation, homogenized with a Polytron homogenizer (Kinematica, Basel, Switzerland), centrifuged at 30,000g for 20 min, and resuspended in PBS. Radioligand binding sites were measured by saturation analysis of specific binding of the ₁-adrenergic receptor antagonist radioligand ¹²⁵I-BE 2254 (20–800 pM). Nonspecific binding was defined as binding in the presence of 10 µM phentolamine. The pharmacological specificity of radioligand binding sites was determined by displacement of ¹²⁵I-BE 2254 (50–70 pM) by prazosin, 5-MU, niguldipine, NE, F18A, and BMY 7378, and data were analyzed using nonlinear regression.

Measurement of [³H]InsP Formation. Accumulation of [³H]inositol phosphates (InsPs) was determined in confluent 96-well plates by a protocol described previously (Hague et al., 2004c). After prelabeling, medium containing [myo-³H]inositol was removed, and 100 µl of Krebs-Ringer bicarbonate buffer (120 mM NaCl, 5.5 mM KCl, 2.5 mM CaCl₂, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 20 mM NaHCO₃, 11 mM glucose, and 0.029 mM Na₂EDTA) containing 10 mM LiCl was gently added to each well. Cells were incubated with or without 100 µM NE for 60 min. For studies using BMY 7378, antagonist was added to cells for 30 min before the addition of agonist. The reaction was stopped by the addition of 100 µl of 20 mM formic acid, and samples were sonicated for 10 s. Samples were subjected to anion exchange chromatography to isolate [³H]InsPs, which were quantified by scintillation counting.

Data Analysis and Statistics. Radioligand binding and [³H]InsP formation data were calculated as means ± S.E.M. and statistical comparisons used GraphPad Prism Software (GraphPad Software Inc., San Diego, CA). Schild plots were calculated according to the method described originally by Arunlakshana and Schild (1959).

	Results

Top Abstract Materials and Methods Results Discussion References

 
_1D-AR Binding Sites Are Undetectable with BMY 7378 in DDT₁MF-2 Cells Expressing _1D-AR Protein. We have shown previously that intracellular _1D-ARs require heterodimerization with _1B-ARs to promote their expression at the cell surface (Hague et al., 2004c). Because DDT₁MF-2 cells endogenously express _1B-ARs at approximately 300 to 400 fmol/mg of protein, we stably transfected these cells with FLAG-_1D-GFP ARs to use as a model system for functional and pharmacological characterization of _1B-/_1D-AR het-erodimers. As expected, confocal microscopy (Fig. 1A) and a luminometer-based cell-surface assay (Fig. 1B) indicated that _1D-ARs were quantitatively expressed at the cell surface. In support of these findings, immunoblotting for FLAG revealed that FLAG-_1D-GFP protein was expressed (Fig. 1C), suggesting that a significant number of _1D-ARs were expressed at the cell surface. Although Western blots are only semiquantitative, careful titration of N-truncated _1D-AR binding site expression with the density of signal on Western blots suggests that this should translate into 600 fmol/mg of protein of _1D-AR binding sites (data not shown). Therefore, we expected to see corresponding increases in the ₁-AR B_max and the appearance of _1D-AR binding sites. However, in saturation binding experiments, we found no significant differences in receptor expression levels between untransfected and _1D-AR expressing DDT₁MF-2 cells (Fig. 1D). In addition, _1D-AR binding sites were undetectable in ¹²⁵I-BE 2254 competition binding experiments using the _1D-AR selective antagonist BMY 7378. Only a single population of _1B-AR low-affinity binding sites consistent with previously observed values at _1B-ARs (Goetz et al., 1995) was observed in both wild-type and _1D-AR transfected cell lines. Thus, our confocal and biochemical data suggested that _1D-ARs were expressed at the plasma membrane after transfection into DDT₁MF-2 cells. However, our findings from radioligand binding experiments suggested that _1D-ARs were not detectable pharmacologically.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Heterologous expression of 1D-ARs with native hamster 1B-ARs in DDT1MF-2 cells. A, confocal imaging of FLAG-1D-GFP ARs stably expressed in DDT1MF-2 cells. Cells were fixed and excited using an argon-neon laser (488 nm) as described under Materials and Methods. B, Cell-surface expression of 1D-ARs in DDT1MF-2 cells. Cell-surface expression of FLAG-1D-GFP ARs was detected using a luminometer-based assay, as described under Materials and Methods. The values for each experiment are represented as the percentage of absorbance over untransfected DDT1MF-2 cells. The data are expressed as mean ± S.E.M. of three independent experiments. C, immunoprecipitation of FLAG-1D-GFP ARs stably expressed in DDT1MF-2 cells. Cells were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG antibodies as described under Materials and Methods. D, 125I-BE 2254 saturation binding analysis of untransfected () or stably transfected DDT1MF-2 cells expressing FLAG-1D-GFP ARs (). Data are expressed as mean ± S.E.M. from three individual experiments performed in duplicate. E, BMY 7378 competition binding analysis of untransfected () or stably transfected DDT1MF-2 cells expressing FLAG-1D-GFP ARs (). Data are expressed as mean ± S.E.M. from four individual experiments performed in duplicate.

_1D-AR Binding Sites Are Undetectable with BMY 7378 in HEK293 Cells Coexpressing _1D-/_1B-ARs. From our data obtained in DDT₁MF-2 cells, we hypothesized that our inability to detect _1D-AR binding sites might be caused by low _1D-AR expression levels, despite the fact that the Western blots suggested that they should be easily detectable. Therefore, we chose to switch to HEK293 cells as a model to characterize _1B-/_1D-AR heterodimers, because in previous studies, we have found that extremely high receptor expression levels can be obtained using this cell line (Uberti et al., 2003; Hague et al., 2004a,c). To create a HEK293 cell line stably coexpressing _1B-/_1D-ARs, we first transfected HA-_1B-ARs in the pREP4 vector and selected with hygromycin until only resistant cells remained. After selection, we confirmed the presence of HA-_1B-ARs by performing ¹²⁵I-BE 2254 competition binding experiments using BMY 7378, which detected a homogenous population of low-affinity binding sites (Fig. 2A). FLAG-_1D-GFP ARs were then stably transfected into HEK293 cells alone or into HEK293 cells stably expressing HA-_1B-ARs. ¹²⁵I-BE 2254 competition binding with BMY 7378 identified a single population of high-affinity BMY 7378 binding sites in HEK293 cells expressing FLAG-_1D-GFP alone. However, similar to our observations in DDT₁MF-2 cells, BMY 7378 detected only a single population of low-affinity binding sites in HEK293 cells coexpressing FLAG-_1D-GFP and HA-_1B-ARs (Fig. 2A). A screen of a panel of ₁-AR-selective antagonists (Table 1) provided no further evidence for the presence of _1D-AR binding sites. The _1A-AR-selective antagonists 5-MU and niguldipine recognized a low-affinity binding site, and the nonselective ligands prazosin, BE 2254, and NE all bound within the range of affinities reported previously. Therefore, to determine whether _1D-ARs were expressed in this cell line using an alternative method, HEK293 cells coexpressing FLAG-_1D-GFP and HA-_1B-ARs were fixed on coverslips and examined using confocal microscopy. As shown in Fig. 2B, FLAG-_1D-GFP ARs were quantitatively expressed at the plasma membrane in this cell line, which was in direct contrast to our radioligand binding data suggesting that _1D-ARs were not expressed. Finally, Western blots from cell lysates were then immunoprecipitated and run on SDS gels and were compared with Western blots from HEK293 cells expressing N-truncated _1D-ARs at approximately 450 fmol/mg of protein (Fig. 2C). The results from our biochemical and confocal studies indicated that _1D-ARs were present when coexpressed with _1B-ARs and should have been forming functional binding sites, yet our pharmacological data indicate that they were not.

View larger version (25K): [in this window] [in a new window]   Fig. 2. BMY 7378 recognizes a single binding site in HEK293 cells coexpressing 1B- and 1D-ARs. A, 125I-BE 2254 competition radioligand binding was used to determine BMY 7378 binding affinities in HEK293 cells expressing FLAG-1D-GFP (), HA-1B (), or coexpressing FLAG-1D-GFP and HA-1B ARs (). Data are the mean of four independent experiments performed in duplicate and are expressed as mean ± S.E.M. B, confocal imaging of fluorescein isothiocyanate fluorescence in HEK293 cells coexpressing FLAG-1D-GFP and HA-1B ARs. Cells were fixed and excited with an argon-neon laser at 488 nm as described under Materials and Methods. C, to semiquantitatively estimate the density of 1D-AR binding sites expected, we performed immunoprecipitation and Western blotting for the FLAG epitope and compared it to N-truncated (Ntr) 1D-ARs, which form binding sites and localize to the cell surface.

View this table: [in this window] [in a new window]   TABLE 1 Log KI values for 1-AR-selective ligands determined from 125I-BE 2254 competition binding studies performed in HEK293 cells stably expressing HA-1B-ARs alone or together with FLAG-1D-GFP ARs

To ensure that _1D-ARs would be expressed at high levels, we used a previously generated HEK293 cell subclone expressing a high density of wild-type (WT) human _1D-AR binding sites (B_max = 920 fmol/mg of protein; data not shown). With this high _1D-AR expression level, we hypothesized that overexpressing _1B-ARs in this cell line would still allow for easy detection of _1D-AR binding sites. WT human _1A-or _1B-ARs in the pREP4 vector were then transfected into the high-expression WT _1D-AR subclone and selected using hygromycin. B_max values were then determined using ¹²⁵I-BE 2254 saturation binding. As shown in Fig. 3A, HEK293 cells transfected with empty pREP4 alone had no significant increase in _1D-AR expression levels (B_max = 1204 fmol/mg of protein), whereas the B_max value in _1A-/_1D-AR-coexpressing cells increased to 1845 fmol/mg of protein (Table 2). It is interesting that the B_max in _1B-/_1D-AR-coexpressing cells was unchanged at 1070 fmol/mg of protein, which was not significantly different from the B_max in HEK293 cells expressing _1D-ARs alone. From these findings, we hypothesized that detection of _1D-AR binding sites with BMY 7378 would be possible in this _1B-/_1D-AR coexpression cell line. As shown in Fig. 3B, ¹²⁵I-BE 2554 competition binding with BMY 7378 revealed the expected result of a heterogeneous population of high- and low-affinity binding sites in _1A-/_1D-AR-coexpressing HEK293 cells and a single population of high-affinity binding sites in _1D-AR-expressing cells with empty pREP4 vector. However, BMY 7378 recognized only a single population of low-affinity binding sites in _1B-/_1D-AR-coexpressing cell lines. These findings have three possible explanations: either 1) all previously expressed _1D-ARs had been replaced with transfected _1B-ARs; 2) _1B-/_1D-ARs were both expressed and formed a heterodimer that has low affinity for BMY 7378; or 3) the presence of _1B-ARs alters the structure of _1D-ARs such that they cannot bind ¹²⁵I-BE 2554.

View larger version (14K): [in this window] [in a new window]   Fig. 3. BMY 7378 recognizes a single population of binding sites in 1B-AR/1D-AR-coexpressing cells. WT 1A-AR and 1B-ARs were stably transfected into HEK293 cells expressing 1D-ARs as described under Materials and Methods. Cell membranes expressing WT 1D-ARs alone () or coexpressed with WT 1A-ARs () or WT 1B-ARs () were prepared and used for 125I-BE 2254 saturation binding (A) and competition binding experiments with BMY 7378 (B). Data are the means of six to nine independent experiments performed in duplicate and are expressed as mean ± S.E.M.

View this table: [in this window] [in a new window]   TABLE 2 Bmax and pKI or pIC50 values determined from 125I-BE 2254 saturation and competition radioligand binding assays in HEK293 cells expressing 1D-ARs

A -T1A Mutant Peptide Reveals Multiple Binding Sites in HEK293 Cells Coexpressing _1B-/_1D-ARs. We have previously characterized a conotoxin peptide -T1A isolated from the sea snail to be an _1B-AR subtype-selective antagonist that acts noncompetitively at the _1B- and competitively at the _1A- and _1D-AR subtypes (Chen et al., 2004). Taken from its differential modes of inhibition at the ₁-AR subtypes, it is likely that -T1A binds to regions of the ₁-ARs other than the conserved catecholamine binding pocket. -T1A is a noncompetitive inhibitor of _1B-ARs but competitively inhibits _1D-ARs (Chen et al., 2004). Figure 4A demonstrates that this peptide is in fact a competitive inhibitor in HEK293 cells coexpressing _1B/_1D-ARs. An alanine mutant of -T1A, F18A, demonstrated significant selectivity between the _1B- and _1D-AR subtypes (20-fold). Therefore, we performed competition binding experiments using F18A in the hope that it would distinguish between _1B-AR and _1D-AR binding sites. As shown in Fig. 4, we found that F18A recognized a single population of low-affinity binding sites in HEK293 cells expressing _1D-ARs and a heterogeneous population of low-affinity binding sites in the _1A-/_1D-AR coexpressing HEK293 cells (Fig. 4). F18A unexpectedly recognized a mixture of high- and low-affinity binding sites in HEK293 cells cotransfected with _1B-/_1D-ARs (Fig. 3C). Ap-proximately 66% of the binding sites were high affinity, with pIC₅₀ values of –9.0, whereas the remaining 34% of the binding sites were low affinity, with pIC₅₀ values of –7.0 (Table 2). Therefore, these data suggest that F18A can recognize multiple binding sites in HEK293 cells cotransfected with _1D-AR and _1B-AR receptor subtypes, whereas BMY 7378 recognizes only a single population of low-affinity binding sites.

View larger version (12K): [in this window] [in a new window]   Fig. 4. The conotoxin peptides -T1A and F18A recognizes multiple binding sites in HEK293 cells coexpressing 1B- and 1D-ARs. WT 1A-AR and 1B-ARs were stably transfected into HEK293 cells expressing 1D-ARs as described under Materials and Methods. A, 125I-BE 2254 saturation binding analysis was performed in the absence () or presence of 30 nM -T1A () in HEK293 membranes coexpressing 1B-/1D-ARs. Data are the means of three independent experiments performed in duplicate and are expressed as mean ± S.E.M. B, cell membranes expressing WT 1D-ARs alone () or coexpressed with WT 1A-ARs () or WT 1B-ARs () were prepared and used for 125I-BE 2254 competition binding experiments with F18A. Data are the means of six to nine independent experiments performed in duplicate and are expressed as mean ± S.E.M.

Coexpression of N-Truncated _1D-ARs with _1B-ARs Reveals _1D-AR Binding Sites. Previous reports from our laboratory (Hague et al., 2004a,c) and others (McCune et al., 2000; Chalothorn et al., 2002) have demonstrated that _1D-ARs are primarily intracellular when expressed alone but can be trafficked to the cell surface upon N-terminal truncation (Hague et al., 2004a) or coexpression with _1B-ARs (Hague et al., 2004c). However, the data shown above suggest that although _1B-ARs can heterodimerize and traffic _1D-ARs to the cell surface, this does not result in an increase in binding site density or _1D-AR binding sites. Therefore, one potential interpretation of these findings is that _1B-/_1D-AR heterodimers form a single receptor complex, resulting in the formation of a novel binding pocket that binds BMY 7378 with low affinity. To test this hypothesis, we coexpressed N-truncated (^1–79) _1D-ARs with _1B-ARs in HEK293 cells. Previous work revealed that N-truncated _1D-ARs are capable of forming heterodimers with _1B-ARs (Uberti et al., 2003) but do not require _1B-AR coexpression for trafficking to the cell surface (Hague et al., 2004a). Thus, we predicted that coexpressing ^1–79_1D-ARs with _1B-ARs may result in the expression of a mixed population of high- and low-affinity BMY 7378 binding sites, because N-truncated _1D-ARs do not depend on _1B-ARs for cell-surface trafficking like the wild-type _1D-ARs do. We created multiple HEK293 cell lines stably expressing ^1–79_1D-GFP ARs and determined their receptor expression levels using ¹²⁵I-BE 2254 saturation binding. As shown in Fig. 5A, approximately 1200 to 1400 fmol/mg of protein of ^1–79 _1D-GFP ARs were expressed in each cell line, with the majority of these receptors expressed at the cell surface, as determined by confocal microscopy (Fig. 5B). HA-_1B-ARs in the pREP4 vector were then transfected into each cell line and selected with hygromycin to produce HEK293 cells coexpressing _1B-/^1–79 _1D-GFP ARs. It is interesting that cell line 1 demonstrated no significant increase in receptor density (B_max = 1126 fmol/mg of protein; Fig. 5A), yet BMY 7378 distinguished a mixed population of high- (58%) and low-affinity (42%) binding sites (Fig. 5B; Table 3). In direct contrast, cell line 2 demonstrated a 2-fold increase in receptor density (B_max = 2902 fmol/mg of protein) (Fig. 5A), but BMY 7378 recognized only a single population of low-affinity binding sites (Fig. 5B; Table 3). Therefore, these findings suggest that at nonsaturating levels of _1B-AR expression, there is a mixed population of ₁-ARs expressed: ^1–79_1D-GFP ARs alone (high-affinity BMY 7378 binding sites), _1B-ARs alone, and _1B-ARs heterodimerized with ^1–79_1D-GFP ARs (low-affinity BMY 7378 binding sites). However, at saturating levels of _1B-AR expression, only low-affinity BMY 7378 binding sites are found, which include _1B-AR and _1B-/^1–79_1D-GFP AR heterodimers.

View larger version (24K): [in this window] [in a new window]   Fig. 5. N-truncated 1D-ARs binding sites are detectable when coexpressed with 1B-ARs. A, GFP-tagged 1–79 1D-ARs were stably transfected into HEK293 cells and were subjected to saturation binding analysis using 125I-BE 2254. Data are the means of four independent experiments performed in duplicate and are expressed as mean ± S.E.M. B, confocal image of HEK293 cells stably expressing GFP-tagged 1–79 1D-ARs. Cells were excited with an argon-neon laser (488 nm) as described under Materials and Methods. C, saturation binding analysis of HEK293 cells coexpressing 1–79 1D-and 1B-ARs. HEK293 cells stably expressing GFP-tagged 1–79 1D-ARs were stably transfected with HA-1B-ARs and subjected to saturation binding analysis using 125I-BE 2254. Cell lines 1 () and 2 () represent separate HA-1B-AR transfections. Data are the means of three independent experiments performed in duplicate and are expressed as mean ± S.E.M. D, HEK293 cells coexpressing 1–79 1D- and 1B-ARs were subjected to 125I-BE 2254 competition binding to determine BMY 7378 affinities. Cell lines 1 () and 2 () represent separate HA-1B-AR transfections and are the same cell lines used in C. Data are the means of three independent experiments performed in duplicate and are expressed as mean ± S.E.M.

View this table: [in this window] [in a new window]   TABLE 3 Bmax and pKI values determined from 125I-BE 2254 saturation and competition radioligand binding assays in HEK293 cells expressing 1-791D-GFP ARs

_1B-/_1D-AR Heterodimers Have Increased Maximal Responses. The data shown above suggest that _1B- and _1D-ARs form heterodimeric complexes that are characterized with low-affinity binding for the _1D-AR-selective antagonist BMY 7378. We next examined the contributions of each ₁-AR subtype to the overall signaling of the _1B-/_1D-AR complex. In previous studies, we found that _1B-/_1D-AR heterodimerization increased the rate of _1D-AR internalization and the maximal levels of intracellular Ca²⁺ mobilization in response to NE stimulation but resulted in only minor increases in maximal PI hydrolysis (Hague et al., 2004c). To further characterize the role of each ₁-AR subtype in the heterodimeric complex, we performed cell-surface assays to determine the rate of _1B-AR internalization. We found that stimulation of _1B-ARs transiently transfected in HEK293 cells resulted in a 40 to 50% loss in the number of cell-surface receptors after 30 min, with no further increase after 60 min (Fig. 6A). Coexpressing _1B-ARs with _1D-ARs resulted in no significant difference in the rate of _1B-AR internalization, suggesting that the _1B-/_1D-AR heterodimer is equally susceptible to agonist-induced endocytosis.

View larger version (16K): [in this window] [in a new window]   Fig. 6. 1B-/1D-AR heterodimers have increased norepinephrine maximal responses. A, coexpression of 1B-/1D-ARs does not effect the internalization parameters of 1B-ARs. Cell-surface expression of FLAG-1B-ARs was determined using a fluorescent luminometer assay as described under Materials and Methods. HEK293 cells expressing FLAG-1B-ARs alone and in combination with HA-1D-ARs were stimulated with 10 µM NE for 30 and 60 min. Data are expressed a mean ± S.E.M. of three experiments performed in triplicate. B, coexpression of 1B-/1D-ARs results in increased NE maximal responses. HEK293 cells expressing 1D-ARs alone (), 1B-ARs alone (), coexpressed 1B-/1D-ARs (), or an equal mixture of cells expressing 1B-ARs and 1D-ARs alone () were incubated with [myo-3H]inositol for 24 h. Cells were then stimulated with increasing concentrations of NE for 1 h and were assayed for [3H]InsP production as described under Materials and Methods. Data are expressed as the percentage of PI hydrolysis, with 100% stimulation equal to the level attained in cells coexpressing 1B-/1D-ARs. Data are the means of three individual experiments performed in duplicate.

_1B- and _1D-ARs Have Distinct Functional Roles within the Heterodimeric Complex. To eliminate any functional responses produced by _1B-AR stimulation, we created HEK293 cell lines stably coexpressing WT _1D-ARs and an _1B-AR mutant missing three amino acids in the N-terminal portion of the third intracellular loop, which is uncoupled from functional responses (12_1B-ARs) but is still capable of promoting cell-surface expression of _1D-ARs (Hague et al., 2004c). Similar to our observations in _1B-/_1D-AR-coexpressing cells, BMY 7378 recognized a single population of low-affinity binding sites in cells expressing _1D-/12_1B-ARs (K_I = –6.27, Fig. 7B), and NE functional responses were significantly greater than those in cells expressing _1D-ARs alone (Fig. 7A). Because 12_1B-ARs do not couple to functional responses (Fig. 7A), we hypothesized that BMY 7378 would inhibit NE functional responses in _1D-/12_1B-AR-coexpressing cells with high affinity. To investigate this, we incubated HEK293 cells stably coexpressing _1D-/12_1B-ARs with increasing concentrations of BMY 7378 for 30 min and generated NE concentration-response curves for InsP formation. Only at high concentrations (1, 3, 10, and 30 µM) did BMY 7378 cause parallel shifts to the right in the NE-concentration curve (Fig. 7C). Schild regression analysis of the data (Fig. 7D) revealed a functional affinity constant of –6.05 ± 0.6 with slope not significantly different from unity. This functional affinity constant for BMY 7378 is characteristic of _1B-AR (6.0) and not _1D-AR (8.5). Therefore, these data provide strong additional evidence that BMY 7378 inhibits NE functional responses at _1B-/_1D-AR heterodimers with low affinity.

View larger version (20K): [in this window] [in a new window]   Fig. 7. 1B- and 1D-ARs form distinct components of a heterodimer signaling complex. A, coexpression of WT 1D-ARs with functionally uncoupled 121B-ARs increases NE maximal responses. HEK293 cells expressing WT 1D-ARs alone (), 121B-ARs alone (), or coexpressed 121B-/1D-ARs () were incubated with [myo-3H]inositol for 24 h. Cells were then stimulated with increasing concentrations of NE for 1 h and assayed for [3H]InsP production as described in Materials and Methods. Data are expressed as the percentage of PI hydrolysis, with 100% stimulation equal to the level attained in cells coexpressing 121B-/1D-ARs. Data are the means of three individual experiments performed in duplicate. B, 125I-BE 2254 competition radioligand binding was used to determine BMY 7378 binding affinity in HEK293 cells coexpressing 121B-/1D-ARs (). Data are the means of three independent experiments performed in duplicate and are expressed as mean ± S.E.M. C, BMY 7378 inhibits NE functional responses in HEK293 cells coexpressing 121B-/1D-ARs with 1B-AR pharmacology. HEK293 cells stably expressing 121B-/1D-ARs were stimulated with increasing concentrations of NE in the absence and presence of 1 µM (), 3 µM (), 10 µM (), and 30 µM () BMY 7378. Data are expressed as the percentage of PI hydrolysis with 100% stimulation equal to the NE maximum and are the mean ± S.E.M. of three individual experiments performed in duplicate. D, Schild plot of BMY 7378 inhibition of NE-stimulated [3H]InsP production in HEK293 cells coexpressing 121B-/1D-ARs.

	Discussion

Top Abstract Materials and Methods Results Discussion References

We were surprised to find that in cell lines stably coexpressing both _1B- and epitope-tagged _1D-ARs, _1D-AR expression could be detected using immunoprecipitation and confocal fluorescence microscopy but could not be detected pharmacologically with the _1D-AR-selective antagonist BMY 7378 in radioligand binding experiments. Comparison of Western blots using the epitope tags suggested that significant numbers of _1D-AR binding sites (500 fmol/mg of protein or greater) should have been present. In addition, no increase in binding-site density was observed in comparison with cells expressing either subtype alone, suggesting that _1B-/_1D-AR heterodimers form a single binding site, or that the presence of _1B-ARs alters _1D-ARs such that they cannot bind ¹²⁵I-BE 2254. However, a mutant of the conotoxin -T1A (F18A) showed biphasic inhibition in cells coexpressing _1B-/_1D-ARs. When coexpressing functionally uncoupled _1B-ARs with full-length WT _1D-ARs, the _1D-AR-selective antagonist BMY 7378 inhibited functional responses to NE with a low affinity, suggesting these two receptors are acting as individual components of a heterodimeric complex. These findings strongly suggest that _1B- and _1D-ARs heterodimerize to form a single functional entity.

One of the most surprising findings of this study was that BMY 7378 was unable to detect _1D-AR binding sites when coexpressed with _1B-ARs, despite the fact that _1D-ARs were detectable with immunoprecipitation and confocal techniques. In fact, when _1B-ARs were stably overexpressed in an HEK293 cell subclone expressing _1D-ARs at very high levels, the number of binding sites did not change, but the pharmacology of BMY 7378 shifted from a single high- to single low-affinity population of sites. This is particularly interesting given that _1D-ARs are largely undetectable with BMY 7378 in most intact tissues (Yang et al., 1997, 1998), despite the fact that _1D-AR mRNA is as widely expressed as the mRNAs for the _1A-AR and _1B-AR subtypes (Rokosh et al., 1994; Alonso-Llamazares et al., 1995; Scofield et al., 1995). For example, in a recent study, mRNA for all three ₁-AR subtypes was detectable in rat submandibular gland cells. However, BMY 7378 detected only a single population of low-affinity binding sites in radioligand binding experiments (Bockman et al., 2004), which is consistent with our findings suggesting that coexpression of _1B- and _1D-ARs results in the masking of high-affinity _1D-AR binding sites. In addition, the affinity of BMY 7378 in inhibiting phenylephrine-mediated contraction was found to be significantly increased in isolated carotid arteries from _1B-AR knockout mice (Deighan et al., 2005), and phenylephrine-stimulated increases in left ventricular-developed pressure were only inhibited by BMY 7378 in _1A-/_1B-AR double knockout mice (Turnbull et al., 2003). These unusual findings could be explained by a model wherein the knockout of _1B-ARs from native tissues results in the unmasking of _1D-AR binding sites with high affinity for BMY 7378, which would be predicted from the results of our cellular studies reported here.

There is an emerging role for dimerization in the biosynthesis and maturation of GPCRs (Bulenger et al., 2005), and it is possible that the expression of the _1B-AR with the _1D-AR relieves a block on the intracellular or post-translational processing of the latter which allows it to be expressed on the cell surface or other aspects of protein maturational processing. Although we do not yet have evidence for such processes, further studies are likely to clarify whether this is important in this interaction.

Rat (Piascik et al., 1995) and mouse (Yamamoto and Koike, 2001) aortas have long been the preferred model system to study _1D-AR functional responses. From our previous studies demonstrating that _1B-AR heterodimerization with _1D-ARs promotes cell-surface expression (Hague et al., 2004c), we expected that _1B-AR knockout mice would have diminished _1D-AR-mediated functional responses. In fact, studies of phenylephrine-stimulated contraction of aorta from _1B-AR knockout mice have given conflicting results. In the original characterization of these mice, aortic contraction was significantly diminished (Cavalli et al., 1997). However, a subsequent study reported that aortic contraction is essentially unaltered in _1B-AR knockout mice (Daly et al., 2002). We reported recently that ₂-ARs can also promote _1D-AR cell-surface expression, and unlike _1D-/_1B-AR heterodimers, they maintain a high affinity for BMY 7378 (Uberti et al., 2005). Therefore, one possibility is that both _1B-ARs and ₂-ARs may contribute to _1D-AR function in mouse aorta.

Accumulating evidence now suggests that each receptor within a GPCR heterodimer is responsible for a particular component of the signaling complex. Several examples of this can be observed in the class III family of GPCRs, including the GABA_B (Jones et al., 1998; Kaupmann et al., 1998) and taste (Nelson et al., 2001) receptors. It is noteworthy that within the GABA_B receptor heterodimer, the GABA_BR2 subunit is responsible for promoting surface expression of the GABA_BR1 (Jones et al., 1998; Kaupmann et al., 1998; Margeta-Mitrovic et al., 2001) by masking an ER retention motif in the GABA_BR1 C-terminal tail (Calver et al., 2000; Margeta-Mitrovic et al., 2000). Once properly assembled, the GABA_BR1 subunit seems to be primarily responsible for agonist binding, whereas the GABA_BR2 subunit couples to G-proteins (Margeta-Mitrovic et al., 2001). It is interesting that we have found that the _1B-/_1D-AR heterodimer is functionally similar to the GABA_B receptor heterodimer. The _1B-AR serves to promote cell-surface expression of the _1D-AR (Hague et al., 2004c), possibly by masking an ER retention motif in the _1D-AR N terminus (Pupo et al., 2003; Hague et al., 2004a; Petrovska et al., 2005). In addition, it seems that within the 12_1B-AR/_1D-AR heterodimer, the 12_1B-AR is primarily responsible for binding ligand, whereas the _1D-AR couples to G protein activation, but whether this is true for wild-type _1B-ARs remains to be determined. Individual receptor subunits acting as distinct components within a heterodimer complex have also been shown previously to occur with heterodimers consisting of H1 histamine and _1B-ARs (Carrillo et al., 2003), ₂-ARs and -opioid receptors (Jordan et al., 2001), ₂-ARs and _2A-ARs (Xu et al., 2003), and ₂-ARs and ₃-ARs (Breit et al., 2004). Taken together, these findings suggest that GPCR heterodimers form functional complexes with distinct pharmacological and signaling properties in which each receptor subunit may be responsible for specific functions. Most of these studies have been done, by necessity, in heterologous expression systems in which receptor density is difficult to control. The functional significance of class I GPCR heterodimers has been demonstrated recently in vivo for opioid receptors using a heterodimer-selective agonist (Waldhoer et al., 2005), consistent with the hypothesis that these complexes occur in native tissues.

The existence of _1B-/_1D-AR heterodimers may seem perplexing, especially because _1B-ARs are functional when expressed alone. We have found that _1B-/_1D-AR heterodimers stimulate greater maximal NE responses relative to _1B-ARs and _1D-AR expressed alone, suggesting that this heterodimer may act as a high-efficacy complex. This is similar to previous findings on _1A-/_1B-AR heterodimerization in which NE responses were 10-fold greater in HEK293 cells coexpressing _1A- and _1B-ARs (Israilova et al., 2004). In addition, a recent study using ₁-AR knockout mice found that _1D-AR and _1D-/_1B-AR knockout mice had a significant decrease in mean arterial blood pressure, whereas _1B-AR knockout mice did not, suggesting that _1D-/_1B-ARs may act cooperatively to regulate blood pressure (Hosoda et al., 2005). Additional evidence for a physiological role of _1B-AR/_1D-AR heterodimers was provided from functional studies of isolated mouse carotid arteries, in which the potency of phenylephrine was significantly decreased in _1D-AR knockout mice yet unchanged in _1B-AR knockout mice (Deighan et al., 2005). These findings raise the possibility that specific heterodimers respond supermaximally to agonist stimulation. Previous studies have reported that the formation of receptor heterodimers results in altered receptor functional characteristics (Breit et al., 2004; Lee et al., 2004). Thus, another possibility is that _1B-/_1D-AR heterodimers are responsible for activating novel transcriptional activators or mitogenic pathways. Future studies are needed to test this hypothesis.

It is becoming increasingly clear that previously unexplained reports of altered pharmacological or functional characteristics of GPCRs may be explained by the formation of heterodimeric complexes. We have found that _1B-/_1D-AR heterodimers mask BMY 7378 high-affinity _1D-AR binding sites, which may explain the inability of BMY 7378 to detect _1D-AR binding sites in native tissues coexpressing _1B- and _1D-ARs. These results raise the possibility that the number of pharmacologically distinct receptor subtypes may be greater than would be predicted by the number of GPCR genes. If true, the use of heterologous systems expressing a single GPCR to screen for novel therapeutics may not accurately reflect the pharmacological complexity of a drug in vivo.

	Acknowledgements

 
We thank Drs. Allan Levey and Howard Rees for help with confocal studies.

	Footnotes

 
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.014985.

ABBREVIATIONS: GPCR, G-protein-coupled receptor; AR, adrenergic receptor; NE, norepinephrine; InsP, inositol phosphate; GFP, green fluorescent protein; HEK, human embryonic kidney; HA, hemagglutinin; ER, endoplasmic reticulum; WT, wild type; PI, phosphatidylinositol; 5-MU, 5-methylurapidil; PBS, phosphate-buffered saline; BE 2254, 2-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone; BMY 7378, 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione.

Address correspondence to: Dr. Kenneth P. Minneman, Department of Pharmacology, Emory Unive