HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 52, 50422-50430, December 27, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/52/50422    most recent M209340200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hanyaloglu, A. C. Articles by Eidne, K. A. Search for Related Content PubMed PubMed Citation Articles by Hanyaloglu, A. C. Articles by Eidne, K. A. Social Bookmarking                      What's this?

Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes

DIFFERENTIAL REGULATION OF -ARRESTINS 1 AND 2^*

Aylin C. Hanyaloglu^§, Ruth M. Seeber, Trudy A. Kohout^¶, Robert J. Lefkowitz^¶, and Karin A. Eidne^§

From the  7TM Receptor Laboratory, Western Australian Institute for Medical Research (WAIMR), University of Western Australia, Centre for Medical Research, and the ^§ Keogh Institute for Medical Research, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Perth, WA 6009, Australia and the ^¶ Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 21170

Received for publication, September 12, 2002, and in revised form, October 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo- and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either -arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only -arrestin 2 could enhance TRHR2 internalization. The preference for -arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a -arrestin 2 knockout or a -arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both -arrestins. The differential -arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and -arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with -arrestins, could also enhance TRHR2/-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the -arrestins and also provides the first evidence that the interactions of TRHRs with -arrestin may be altered by hetero-oligomer formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G-protein-coupled receptor (GPCR)¹ function is underpinned by the formation of protein-protein interactions and complexes that are dynamically regulated by a variety of stimuli. These molecular interactions are involved in receptor recognition, activation, and desensitization and are targets for the majority of current therapeutic compounds utilized to treat a variety of disorders. More recently, the escalating body of evidence for GPCR oligomerization also adds another level of complexity in understanding how GPCRs are activated and signal and traffick in the cell. GPCR hetero-oligomerization may explain the diverse physiological responses obtained from a single ligand. Indeed, a number of GPCR subtypes such as the somatostatin, opioid, angiotensin, and -adrenergic receptors have been shown to form hetero-oligomers that result in a receptor unit with either altered ligand specificity, signaling, or internalization rates (1-5). GPCR activation is also regulated by receptor-protein interactions involved in desensitization and internalization. For the majority of GPCRs, the mechanism of internalization is via the -arrestin and dynamin-dependent pathway where agonist-activated phosphorylated receptors interact with the -arrestins (-arrestin 1 and 2). Receptors are then targeted to clathrin-coated pits by -arrestins and subsequently undergo endocytosis (6-9). How oligomerization (homo versus hetero) impacts on receptor desensitization, specifically by its interactions with intracellular proteins, has not been explored.

Thyrotropin-releasing hormone (TRH) is an important hypothalamic neuropeptide with well-documented roles in controlling production of TSH and prolactin from the anterior pituitary gland mediated by TRH receptor 1 (TRHR1). TRH is also known to have extrapituitary actions in the brain, spinal cord, cardiovascular, and gastrointestinal systems (10, 11). The cloning of a second receptor for TRH (TRHR2) from rat brain and spinal cord provided a possible explanation for certain neurotransmitter actions of TRH, in particular the nociceptive and spinal cord regenerative actions (12-14). The two TRH receptor subtypes are ~50% homologous, and they exhibit similar binding affinities for TRH (12-14) and activate the same signaling pathways, although TRHR2 exhibits higher basal signaling activity (14-16). TRHR1 and 2 are expressed in distinct compartments of the brain and spinal cord (12, 14, 17, 18), although areas have been defined where both receptor subtypes are found (12, 14). This raises the question regarding the mechanism of selectivity and receptor regulation used when two individual receptors, or a putative TRHR hetero-oligomeric unit, are activated by one ligand to produce differential cellular responses.

In contrast to the extensive studies carried out on both TRHR1 function and regulation by our group and others (19-30), only limited studies have been carried out on TRHR2. It has been established that TRHR1 internalizes via clathrin-coated pits and follows a -arrestin- and dynamin-dependent pathway (22, 23, 26, 31). However, details regarding the mechanism of internalization for the TRHR2 are unknown. We have also demonstrated homo-oligomerization of the TRHR1 using a biophysical method, bioluminescence resonance energy transfer (BRET) (30); however, it is not known if TRHR2 undergoes homo-oligomerization, nor if TRHR subtypes have the potential to hetero-oligomerize. BRET represents a powerful technique to study protein-protein interactions in living cells and has been used to provide evidence for homo and heterodimerization of other GPCRs (4, 32-37). In the present study, we show that TRHR subtypes could form both homo-oligomers and hetero-oligomers in living mammalian cells. We also investigated how each of the two TRHR subtypes utilize the arrestins to promote internalization and found that TRHR subtype internalization was differentially regulated by -arrestin 1 and 2, in that TRHR2 internalization was insensitive to -arrestin 1. Additionally, formation of TRHR hetero-oligomers affects internalization kinetics, and through using BRET and confocal microscopy this may be a consequence of altered interactions with -arrestin. Our findings suggest that TRHR oligomerization results in the creation of novel receptor units that can be differentially regulated by -arrestins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- TRH was obtained from Peninsula Laboratories Europe Ltd (Merseyside, UK). [[³H]Me-His²]TRH was supplied by PerkinElmer Life Sciences (Boston).

Eukaryotic Expression Constructs-- Rat TRHR1 coding region was PCR-amplified from a previously described clone (38) and inserted into pcDNA3 vector. The TRHR1/Rluc and TRHR1/EYFP constructs have been previously described (30). TRHR2/Rluc and TRHR2/EYFP were created by PCR amplification from the rat TRHR2 cDNA (12) (provided by P. Walker, Astra Medical Research Center, Montreal, Canada). The fragment was then cloned in-frame into the pRluc and pEYFP vectors as previously described (30). TRHR1 C335Stop was created by PCR amplification from the rat TRHR1 coding region replacing cysteine 335 with a stop codon. The TRHR1 C335/Rluc was PCR-amplified from amino acids 1 to 334 of the rat TRHR1-coding region and cloned in-frame into the pRluc vector (30). The rat gonadotropin-releasing hormone receptor (GnRHR) was PCR-amplified from a previously described clone (39) and inserted into pcDNA3. The GnRHR/EYFP construct has been previously described (30). Human orexin receptor 2 cDNA was provided by M. Yanagisawa (University of Texas Southwestern Medical Center at Dallas). The -arrestin 1 and -arrestin 2 cDNA were provided by J. Benovic (Kimmel Cancer Institute, Philadelphia). -arrestin 1/EYFP has previously been described (30). The -arrestin 2/EYFP fusion construct was provided by M. Bouvier (University of Montreal, Canada) and is also previously described (32). Wild-type dynamin and K44A dynamin were provided by M. Caron (Duke University Medical Center, North Carolina). Sequences of all cDNA clones were verified using Dye Deoxy sequencing and an ABI 373 sequencer (PE Applied Biosystems).

Cell Culture and Transfection-- HEK293, COS-1 cells (American Type Culture Collection), and MEFs from wild type and -arrestin knockout (-arr-KO) animals; -arr1-KO (-arr1/; -arr2+/+), -arr2-KO (-arr1+/+; -arr2/), -arr1/2-KO (-arr1/; -arr2/) (40) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, glutamine (0.3 mg/ml), and penicillin/streptomycin (100 units/ml) (Invitrogen, Melbourne, Australia) at 37 °C in 5% CO₂. Transient transfections were performed with SuperFect or PolyFect (Qiagen, Australia), and cells were assayed 48 h post-transfection.

Confocal Imaging-- Transfected HEK293 cells were plated onto poly-L-lysine coated 8-well chamber slides 24 h after transfection. Treatments were carried out 48 h post-transfection and cells fixed in 4% paraformaldehyde. Cells were exposed to a 488-nm laser light and examined using a BioRad Confocal Laser Microscope under an oil immersion ×60 objective with light filtered in the green channel from 500 to 550 nm.

Whole Cell Receptor Binding Assay-- Dose displacement receptor binding assays were performed on transiently transfected COS-1 cells expressing untagged and tagged TRHR expression constructs in 24-well plates. Briefly, cells were incubated for 120 min at 4 °C in assay buffer (HEPES buffered Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin) with [[³H]Me-His²]TRH (PerkinElmer Life Sciences) with or without unlabeled agonist (10⁵ to 10¹¹ M). The cells were then washed and solubilized in 0.2 M NaOH/1%SDS and the radioactivity counted. Saturation binding assays were carried out using [[³H]Me-His²]TRH (10 nM-30 pM), nonspecific binding was determined in the presence of unlabeled agonist (1 µM). B_max values were determined by non-linear regression using PRISM (GraphPad Software, Inc) and expressed as receptor sites/cell using Avogadro's number.

Flow Cytometry-- Flow cytometry was used to monitor expression of either EYFP-tagged receptors or -arrestins. Transfected cells were detached with 0.05% trypsin/phosphate-buffered saline and resuspended in 3% fetal calf serum/phosphate-buffered saline and measured in a Becton Dickinson FACSCalibur^TM (San Jose, CA). Cells were gated to exclude dead cells and debris, and analysis was typically carried out on 10,000 events. Data were analyzed using CellQuest.

Receptor Internalization Assay-- Receptor internalization assays were performed as previously described (25).

BRET Assay-- COS-1 cells were detached with 0.05% trypsin/phosphate-buffered saline and washed twice in phosphate-buffered saline. Approximately 50,000 cells per well were incubated in the presence or absence of TRH (1 µM final concentration) for the specified time period at 37 °C. The coelenterazine (h form) (Molecular Probes) was added to a final concentration of 5 µM, and readings were collected immediately following this addition. Repeated readings were taken for at least 5-10 min using a custom-designed BRET instrument (Berthold, Australia) that allows sequential integration of the signals detected in the 440-500 nm and 510-590 nm windows. The BRET ratios for the co-expression of the Rluc and EYFP constructs were normalized against the BRET ratio for the Rluc expression construct alone. For receptor/-arrestin experiments the data are represented as the agonist-dependent increase of the normalized BRET ratio over untreated cells. The BRET ratio is defined as [(emission at 510-590)  (emission at 440-500) × cf]/(emission at 440-500), where cf corresponds to (emission at 510-590/emission at 440-500) for the Rluc construct expressed alone in the same experiment (30, 32).

Total Inositol Phosphate (IP) Assays-- Total IPs were extracted and separated as described previously (41).

Statistical Analysis-- Statistical significance was determined using the Student's t test. Differences are considered significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Comparison of the Internalization Rates of TRHR Subtypes-- The internalization rates of TRHR subtypes were compared following their expression in either COS-1 or HEK293 cells (Fig. 1). While expression levels for TRHR1 were ~2.3-fold less than that observed for TRHR2 (B_max values: TRHR1 287,902 receptor sites/cell; TRHR2 668,564 receptor sites/cell), the maximal level of TRHR1 internalization was greater than that measured for TRHR2 (Fig. 1A), and measurements of half-time rates of internalization (t_1/2) showed a much faster rate for TRHR1 when compared with TRHR2 in both cell lines (Fig. 1B). The relatively shorter t_1/2 values of both TRHR subtypes observed in HEK293 cells may be due to the higher endogenous levels of -arrestins in these cells compared with COS cells, known to express only low levels of -arrestins (42). By varying the expression of each TRHR subtype, the rate, or extent, of receptor internalization was not altered (data not shown). Therefore, TRHR subtypes 1 and 2 demonstrated significantly different internalization kinetics.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Comparison of TRH-stimulated internalization rates of TRHR1 and TRHR2. A, COS-1 or HEK293 cells were transiently transfected with TRHR1 or TRHR2, plated into 24-well plates, and assayed 48 h post-transfection. Internalization assays were carried out after TRH stimulation (5-120 min). Each time point was carried out in duplicate and was repeated at least three times. Results shown are mean ± S.E. and are representative of a single experiment. B, half-time values of internalization (t1/2, min) for TRHR1 and TRHR2 in COS-1 and HEK293 cells. Results shown are mean ± S.E. of three separate experiments.

TRHR2 Internalization Is Via a Clathrin-mediated Pathway-- It has previously been shown that TRHR1 is internalized via a clathrin-mediated pathway (23, 26), a process which can be inhibited by either a dominant negative dynamin mutant (dynamin K44A) (43-45), or the presence of hypertonic medium (0.4 M sucrose), which is known to disrupt the formation of clathrin cages (46). Therefore, in order to determine if TRHR2 is internalized via a similar pathway, we investigated the effect of both dynamin K44A and sucrose on TRHR2 internalization. Co-expression of dynamin K44A, but not wild type dynamin, with TRHR2 inhibited internalization by ~50% in both HEK293 (Fig. 2) and COS-1 cells (data not shown). Preincubating cells with sucrose for 20 min prior to and during stimulation with TRH (1 h) also resulted in an almost complete inhibition of TRHR2 internalization (Fig. 2). These results suggest that similar to TRHR1, internalization of TRHR2 is via a clathrin-mediated process.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   TRHR2 internalization is via a clathrin-mediated pathway. HEK293 cells were transfected with TRHR1 or TRHR2 with either pcDNA3 (control), wild type (WT), dynamin, or dynamin mutant K44A. Internalization assays were carried out after 1 h of treatment with TRH. Cells transfected with TRHR1 or TRHR2 were treated with sucrose (0.4 M for 20 min) or vehicle prior to and during TRH stimulation (1 h). Assays were carried out at least three times, and the results shown are mean ± S.E.

Differential Regulation of TRHR Subtype Internalization by -Arrestins-- As both -arrestin 1 and 2 have been shown to regulate TRHR1 internalization (25, 26), their role in TRHR subtype internalization was evaluated. Internalization assays were carried out on transfected COS-1 cells following TRH treatment (5-120 min). -arrestin 1 significantly promoted TRHR1 internalization, increasing the receptor intracellular pool at 120 min from ~28 to 46% (Fig. 3A) as previously reported (25, 26), while -arrestin 2 promoted TRHR1 internalization to a similar extent (Fig. 3B). Strikingly, only co-expression of -arrestin 2 significantly increased levels of TRHR2 internalization from 14 to 31% at 120 min (Fig. 3D), while -arrestin 1 had only a minimal effect on the levels of internalized TRHR2 (Fig. 3C). The -arrestin 1-independence of TRHR2 was not due to the effect of -arrestin 1 on TRHR2 expression since flow cytometry analysis with EYFP-tagged TRHRs transfected in the absence or presence of -arrestin 1 or 2 showed no significant difference upon co-expression of either -arrestin isoform (percentage of TRHR1 expression only; TRHR1 and -arrestin 1, 138 + 35.8%, TRHR1 and -arrestin 2, 90 ± 8%; percentage of TRHR2 expression only; TRHR2 and -arrestin 1, 138 ± 33%, TRHR2 and -arrestin 2, 103 ± 11%). Therefore, these results demonstrate a differential regulation of TRHR subtype internalization by -arrestins isoforms.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Differential effect of -arrestin 1 and -arrestin 2 on TRHR subtype internalization. COS-1 cells were transiently transfected with TRHR1 (A and B) or TRHR2 (C and D) with pcDNA3 (), -arr 1 (A and C) (), or -arr 2 (B and D) (). Cells were seeded into 24-well plates and assayed 48-h post-transfection. Internalization assays were carried out after TRH stimulation (5-120 min). Each time point was carried out in duplicate and was repeated at least three times. Results shown are the mean ± S.E. and are representative of a single experiment.

Effect of Reduced Levels of -Arrestins on TRHR Subtype Internalization-- Assessment of the functional roles of -arrestin on TRHR subtype trafficking is greatly facilitated by the availability of MEF cell lines derived from -arrestin knockouts, -arr1-KO, -arr2-KO, and -arr1/2-KO (40). TRHR1 displayed reduced TRH-stimulated internalization in the -arr1-KO and -arr2-KO cell lines compared with wild type MEFs (p < 0.05; Fig. 4, WT). However, while this decrease has previously been attributed to lower levels of each remaining -arrestin (40), the -arr1/2-KO lines exhibited a dramatic (87%) reduction in TRH-induced TRHR1 internalization (p < 0.01, Fig. 4). These results support our previous data (Fig. 3 and Refs. 25 and 26) that the TRHR1 can internalize with either -arrestin 1 or 2. However, a different pattern was observed for TRHR2, TRH-induced internalization of TRHR2 was not significantly different in the -arr1-KO cells compared with WT MEFs, while TRHR2 internalization was impaired in both the -arr2-KO and the -arr1/2-KO cell lines, (90 and 85% of WT MEFs respectively) (Fig. 4). Thus, these data support our observation that -arrestin 2, and not -arrestin 1, is responsible for regulating TRH-induced TRHR2 internalization.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Differential agonist-induced TRHR subtype internalization in -arrestin knockout cell lines. MEF cell lines derived from wild type (WT), -arrestin 1 knockout (-arr1-KO), -arrestin 2 knockout (-arr2-KO), and -arrestin 1/2 knockout (-arr1/2-KO) were transfected with either TRHR1 or TRHR2. Internalization assays were carried out after 1 h of treatment with TRH. Each time point was carried out in duplicate, and results shown are the mean ± S.E. from three experiments. , p < 0.05, , p < 0.01 as compared with TRHR1 expressed in WT MEFs. **, p < 0.01 as compared with TRHR2 expressed in WT MEFs.

Differential Translocation of GFP-tagged -Arrestins Following TRH Stimulation-- Because of the differential ability of -arrestins to promote TRHR subtype internalization, we next examined the intracellular localization of GFP-tagged -arrestins following TRH stimulation. In HEK293 cells expressing TRHR1, addition of TRH caused a rapid translocation (within 90 s) of both -arrestin 1/GFP (Fig. 5A) and -arrestin 2/GFP (Fig. 5B) from the cytoplasm to the cell surface. However, in cells expressing TRHR2, only -arrestin 2/GFP (Fig. 5B), and not -arrestin 1/GFP (Fig. 5A), translocated to the cell surface following TRH treatment, supporting the internalization data in Figs. 3 and 4. It has been previously demonstrated that TRHR1 co-internalizes with -arrestin 1 into endocytotic vesicles following prolonged treatments with TRH (15 min), causing a redistribution of -arrestin 1/GFP into a vesicular pattern (22). Our data at 15 min confirmed this finding for TRHR1 for both -arrestin 1/GFP and -arrestin 2/GFP (Fig. 5, A and B). In direct contrast, longer exposure of TRH to cells co-expressing TRHR2 and -arrestin 1/GFP did not demonstrate any change in the distribution of -arrestin 1 (Fig. 5A). In addition, in cells expressing TRHR2, -arrestin 2/GFP remained at the cell surface (Fig. 5B), even though a significant amount of TRHR2 had been internalized following prolonged TRH stimulation (15 min, Fig. 3). Thus, in addition to their differential utilization of -arrestins to promote internalization, TRHRs also differ in their regulation of the trafficking of translocated -arrestins.

View larger version (71K): [in this window] [in a new window]   Fig. 5.   -arrestin/GFP trafficking in cells expressing TRHR1 and TRHR2. Visualization of the agonist-dependent redistribution of -arrestin 1/GFP (A) and -arrestin 2/GFP (B) was assessed using confocal microscopy. HEK293 cells were transfected with either -arrestin 1/GFP or -arrestin 2/GFP with either TRHR1 or TRHR2 as indicated and treated with TRH for 90 s or 15 min.

Direct Interaction of TRHRs with -Arrestins in Live Cells-- Visualization of -arrestin/GFP trafficking using confocal microscopy does not measure the direct association of TRHR/-arrestin, nor does it have the ability to detect low level protein interactions. Therefore, we employed the highly sensitive BRET method for the quantitative analysis of TRHR/-arrestin interactions in living cells in order to assess direct interactions between TRHR2 and -arrestin 1. Potential interacting proteins are either fused to the donor Rluc or acceptor EYFP molecules. If the Rluc and EYFP moieties are ~100 Å or less apart, energy resulting from the rapid oxidation of a cell permeable substrate, coelenterazine, by Rluc, will transfer to EYFP, which in turn emits fluorescence. TRHR1 and TRHR2 were tagged at their C terminus with either Rluc or EYFP. Addition of the tags to each of the receptors (Table I and Ref. 30) does not significantly impair either binding or ligand-induced internalization properties (Table I), although we also observed that the maximal internalization levels of TRHR1/EYFP were slightly reduced as previously published (Table I and Ref. 30). Both -arrestin 1 and 2 were tagged with EYFP and have been previously characterized (30, 32).

View this table: [in this window] [in a new window]   Table I Functional characterization of tagged BRET fusion TRHR constructs IC50 values for WT and BRET fusion TRHR constructs were measured by whole cell displacement binding assays carried out in COS-1 cells expressing either WT TRHRs or TRHR fusion constructs in the presence of increasing amounts of unlabeled agonist (1011  105 M). Results shown are the mean ± S.E. of three independent experiments. Internalization assays were performed in HEK293 cells expressing either WT or BRET fusion constructs following TRH stimulation (106 M, 60 min). Assays were carried out at least three times, and results shown as mean ± S.E. and are representative of a single experiment.

HEK293 cells were transiently transfected with TRHR1/Rluc or TRHR2/Rluc and either pcDNA3, -arrestin 1/EYFP or -arrestin 2/EYFP. Following the addition of coelenterazine, readings were taken immediately and a bioluminescent signal was emitted in both the 440-500 nm and 510-590 nm filter windows in cells co-expressing either TRHR1/Rluc or TRHR2/Rluc with pcDNA3. To quantitate the BRET signal, the ratio of light emitted in the 510-590-nm window over that emitted in the 440-510-nm window was determined as described under "Experimental Procedures."

Following exposure to TRH, cells expressing TRHR1/Rluc and either -arrestin 1/EYFP or -arrestin 2/EYFP showed a similar agonist-dependent increase in the BRET ratio over untreated cells (0.25 and 0.27, respectively; Fig. 6). In addition, a control mutant (TRHR1 C335Stop), which is able to bind ligand and signal similarly to wild type TRHR1 (data not shown), but is unable to recruit -arrestins or undergo ligand-stimulated internalization (27, 31, 47), did not interact with either -arrestin 1 or 2 with BRET (Fig. 6). These results therefore demonstrated the validity of the BRET methodology.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Direct measurement of the interaction between -arrestins and TRHR subtypes using BRET. BRET was measured in HEK293 cells transfected with TRHR1/Rluc, TRHR1 C335/Rluc, or TRHR2/Rluc and either pcDNA3, -arrestin 1/EYFP or -arrestin 2/EYFP. Cells were incubated with 5 µM coelenterazine, and BRET readings were taken from 0-10 min. TRH (106 M final concentration) was added to the same cells and readings were continued for an additional 10 min. Results shown are the mean ± S.E. increase in the BRET ratio obtained for treated over untreated cells, of three separate experiments.

Addition of TRH to cells expressing TRHR2/Rluc and -arrestin 2/EYFP resulted in a higher agonist-dependent increase in the BRET ratio than in cells expressing TRHR2/Rluc and -arrestin 1/EYFP, with values of 0.19 and 0.04, respectively (Fig. 6). Therefore, the more sensitive BRET technique detected a small interaction occurring between TRHR2 and -arrestin 1 that was not observed previously by means of confocal microscopy (Fig. 5).

Homo- and Hetero-oligomerization of TRHR Subtypes-- Using BRET, we have recently shown that TRHR1 undergoes constitutive homo-oligomerization, and the BRET signal is further modulated upon addition of TRH (30). Therefore, we wanted to establish if TRHR2 is also able to undergo homo-oligomerization in the presence and absence of ligand. In addition, we wished to examine the possibility that TRHR1 and TRHR2 can form hetero-oligomers, particularly since they are co-expressed in certain sites in the brain and could thus form another level of physiological regulation by TRH.

Measurements of receptor homo- and hetero-oligomer formation were performed by means of BRET. Cells co-expressing TRHR1/Rluc and TRHR1/EYFP showed an increase in the BRET ratio (0.2) relative to that for TRHR1/Rluc expressed alone (Fig. 7A), and addition of TRH resulted in a further increase in BRET ratio (0.36). These results are similar to our previously published data (30). Likewise, TRHR2 displayed constitutive and agonist-dependent receptor homo-oligomerization, with ratios increasing from 0.17 in untreated cells, to 0.27 in treated cells (Fig. 7A). Therefore, these results indicate that both TRHR subtypes are able to form homo-oligomers.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Homo- and hetero-oligomerization of TRHR1 and TRHR2. A, COS-1 cells were transfected as indicated. BRET assays were performed 48 h post-transfection. For untreated samples, 5 µM coelenterazine was added to cells, and repeated readings taken immediately. For treated samples, cells were incubated with 1 µM TRH for 20 min prior to addition of coelenterazine. Assays were performed at least three times, and results show mean ± S.E. of three independent experiments. B, specificity of TRHR subtype hetero-oligomerization was demonstrated as follow: COS-1 cells transfected with TRHR1/Rluc and TRHR2/EYFP were also transfected with either pcDNA3, untagged TRHR1, TRHR2, or GnRHR. Assays were performed at least three times, and results show mean ± S.E. and are representative of a single experiment.

The ability of TRHR1 and TRHR2 to form hetero-complexes was also examined. Co-expression of TRHR1/Rluc with TRHR2/EYFP resulted in a BRET ratio of 0.23 and agonist treatment resulted in a further increase to 0.37 (Fig. 7A). Similar results were obtained with reciprocally tagged receptors (TRHR2/Rluc and TRHR1/EYFP) indicating that the BRET signal did not depend on the position of the tags relative to the receptors (not shown). Specificity of the interactions was demonstrated by the lack of a significant BRET signal detected between TRHR1/Rluc or TRHR2/Rluc with GnRHR/EYFP (Fig. 7A), or with several other EYFP-tagged GPCRs at similar expression levels (data not shown). These results therefore indicate that TRHR1 and TRHR2 formed constitutive hetero-oligomeric complexes in living cells and that treatment with agonist resulted in an increase in the BRET ratio.

Further experiments to determine the specificity of the TRHR hetero-oligomerization interaction was carried out with TRHR1 and TRHR2 BRET-tagged constructs expressed in either the presence or absence of untagged receptors (Fig. 7B). Both untagged TRHR1 or TRHR2 were found to compete with and thus markedly reduce the constitutive TRHR1/Rluc and TRHR2/EYFP BRET signal (Fig. 7B), although co-expression of another untagged GPCR, the GnRHR, at similar expression levels, did not significantly reduce the BRET signal obtained between the tagged TRHR1 and TRHR2 (Fig. 7B). This suggests that the constitutive BRET signal observed between tagged TRHR1 and TRHR2 was due to a specific interaction.

Functional Analysis of TRHR Hetero-oligomerization-- It has been shown for other GPCRs that heterodimerization can result in altered pharmacological and functional properties distinct from each of the individual receptors (48-51). Therefore, we wanted to determine if co-expression of TRHR1 and TRHR2 resulted in any alterations in receptor function such as ligand binding, TRH-stimulated IP production and ligand-induced internalization.

TRH displacement binding assays were performed in COS-1 cells with concentrations of unlabeled ligand ranging from 10⁵ M to 10¹¹ M. The IC₅₀ values for TRH binding were similar for each TRHR subtype expressed individually, therefore co-expression of TRHR1 and TRHR2 did not significantly affect ligand binding (Fig. 8A).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Functional analysis of TRHR1 and TRHR2 homo and hetero-oligomers. A, displacement TRH binding assay in HEK293 cells with unlabeled ligand (105-1011 M). B, agonist-induced total IP accumulation was measured in HEK293 cells transiently expressing TRHR1, TRHR2, TRHR, or TRHR1/TRHR2. Total IPs were extracted and measured after treatment with TRH (1012 M - 106 M, 45 min) as described under "Experimental Procedures." Results shown are the mean ± S.E. of triplicate observations from a single representative experiment. Nonlinear regression analyses and curve fitting were performed with PRISM software (GraphPad Software, Inc.). C, internalization assays were performed in transfected HEK293 cells following TRH treatment (5-120 min). All assays were performed in duplicate or triplicate in at least three independent experiments. Results shown are mean ± S.E. of a single representative experiment. Tabulated half-time values (t1/2, min) were mean ± S.E. of three independent experiments. , significantly different from TRHR1, p < 0.05; *, significantly different from TRHR2, p < 0.05.

Following TRH treatment, IP production was measured in cells expressing each of the TRHRs individually or together (Fig. 8B). TRHR2 had higher basal IP signaling compared with TRHR1, as previously reported (14-16), while maximal values observed were similar. Co-expression of TRHR1 and TRHR2 exhibited intermediate basal signaling levels when compared with each of the two subtypes expressed individually, although maximal stimulation was similar. EC₅₀ values of cells expressing both TRHR subtypes were not significantly different to those cells expressing either TRHR1 or TRHR2 (TRHR1 4.9 + 2.1 nM; TRHR2 1.3 + 0.4 nM; TRHR1/TRHR2 2.3 + 0.8 nM).

Measurement of TRH-induced internalization in HEK293 cells co-expressing TRHR1 and TRHR2 revealed a significantly altered internalization rate (p < 0.05) compared with each of the TRHRs expressed individually (Fig. 8C). Therefore, while these results demonstrated no significant difference in ligand binding and an intermediate level of IP signaling, significant alterations in receptor internalization rates occurred when TRHR hetero-oligomers were formed.

TRHR Hetero-oligomerization Alters the Interaction Between TRHR2 and -Arrestin 1-- We wished to examine whether altered trafficking of TRHR hetero-oligomers versus homo-oligomers might have functional implications. Our observation that TRHR subtype trafficking can be differentially regulated by the different -arrestins may provide us with a strategy for addressing the mechanism underlying the altered kinetics of TRHR hetero-oligomers. By means of BRET, we can monitor the direct interactions between arrestin isoforms and receptor complexes and thus determine whether the TRHR2/-arrestin 1 BRET interaction could be influenced by hetero-oligomerization with TRHR1.

In cells co-expressing TRHR1/Rluc and -arrestin 1/EYFP, transfection of untagged TRHR1 or TRHR2 had no significant effect on the BRET signal obtained with (Fig. 9A). However, the small BRET signal obtained with cells co-expressing TRHR2/Rluc and -arrestin 1/EYFP (Figs. 6 and 9B) increased significantly (3-fold) when transfected with untagged TRHR1 (p < 0.01) but not untagged TRHR2 (Fig. 9B).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Modulation of the BRET interaction between tagged TRHR2 and -arrestin 1 by untagged TRHRs. HEK293 cells were transfected with either TRHR1/Rluc with -arrestin 1/EYFP (A) or TRHR2/Rluc with -arrestin 1/EYFP (B). In addition, pcDNA3, untagged TRHR1, TRHR2, or TRHR1 C335stop were co-expressed. Cells were incubated with 5 µM coelenterazine, and BRET readings were taken from 0 to 10 min. TRH was added at 106 M final concentration to the same cells, and readings were continued for an additional 10 min. Results shown are the mean ± S.E. increase in the BRET ratio obtained for treated over untreated cells from three separate experiments. *, p < 0.05 and **, p < 0.01 as compared with TRHR2/Rluc + -arrestin 1/EYFP co-transfected with pcDNA3.

As TRHR1 can form hetero-oligomers with TRHR2, the promotion in the TRHR2/-arrestin 1 BRET signal could be explained by the ability of TRHR1 to efficiently bind to -arrestin 1 (Figs. 6 and 9A) and that the close proximity of TRHRs within the hetero-oligomer permits a greater degree of energy transfer from TRHR2/Rluc to -arrestin 1/EYFP. To address this possibility, the truncated receptor, TRHR1 C335Stop, which is unable to interact directly with -arrestin 1 (Fig. 6), was co-expressed with either of the Rluc-tagged TRHRs and EYFP-tagged -arrestin 1. The TRHR1 C335Stop was also able to significantly promote the TRHR2/Rluc and -arrestin 1/EYFP BRET signal (p < 0.05) to similar levels observed with untagged wild type TRHR1 (Fig. 9B) but had no effect on the BRET signal obtained between TRHR1/Rluc and -arrestin 1/EYFP. This indicates that the potentiation of TRHR2/-arrestin 1 by TRHR1 C335Stop was not because -arrestin 1 had been brought into closer proximity with TRHR2 but was due to its interaction with TRHR1 (Fig. 9A). Overall, these results suggest that formation of TRHR1/TRHR2 oligomer results in altered -arrestin 1 binding to the TRHR2.

TRHR Hetero-oligomers Have Altered Trafficking of -Arrestin 1/GFP-- As the BRET results indicated that an increased interaction occurs between TRHR2 and -arrestin 1 in the presence of either TRHR1 or the -arrestin-independent receptor TRHR1C335Stop, assessment of altered -arrestin 1 trafficking was also carried out by confocal microscopy. HEK293 cells were transfected with -arrestin 1/GFP and TRHRs (TRHR1, TRHR2, TRHR1 C335Stop) expressed individually or together, as indicated in Fig. 10 and cells were monitored for the localization of -arrestin 1/GFP following TRH treatment. Cells co-expressing TRHR1 and TRHR2 showed a rapid translocation of -arrestin 1/GFP to the cell surface, which then remained localized at the plasma membrane following longer agonist treatment (Fig. 10). This is in contrast to cells expressing TRHR1 only (Fig. 5) where -arrestin 1/GFP redistributes into vesicles following longer stimulation. Interestingly, in cells co-expressing TRHR2 and TRHR1 C335Stop, neither of which causes a visible translocation of -arrestin 1/GFP when expressed individually (Figs. 5 and 10), we observed a rapid agonist-dependent redistribution to the cell membrane, which remained localized following longer treatments with TRH (Fig. 10). TRHR1 C335Stop had no effect on TRHR1 trafficking of -arrestin 1/GFP. Therefore, these results support the BRET data that TRHR2 can form hetero-oligomers with either TRHR1 or TRHR1 C335Stop and that this interaction enables TRHR2 to translocate -arrestin 1. In addition, formation of hetero-oligomers alters the subsequent ability of -arrestin 1 to redistribute into intracellular vesicles when compared with TRHR homo-oligomers.

View larger version (70K): [in this window] [in a new window]   Fig. 10.   TRHR hetero-oligomers alter -arrestin 1/GFP trafficking. Visualization of the agonist-dependent redistribution of -arrestin 1/GFP was assessed using confocal microscopy. HEK293 cells were transfected with -arrestin 1/GFP and either TRHR1, TRHR2, or TRHR1 C335Stop as indicated. Cells were untreated or treated with TRH for 90 s or 15 min.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is not yet understood how different GPCR subtypes, which are activated by the same ligand, induce differential cellular responses. Multiple receptor signals could be achieved by differential regulation of receptor subtypes by intracellular proteins or formation of new functional units through GPCR hetero-oligomerization. Ultimately, these effects would alter receptor trafficking properties and cell responsiveness to the agonist, thus serving a crucial role in generating the diversity of responses required for regulating cellular function in living systems.

The TRHR subtypes (TRHR1 and TRHR2) are activated by the same ligand and are expressed in both unique and overlapping brain regions (14, 18). To analyze subtype-specific differences in TRH-stimulated receptor internalization, we performed comparative studies of the cellular processes involved in receptor internalization by examining the role of -arrestin isoforms. In addition, we explored the potential of TRHR1 and TRHR2 to hetero-oligomerize and analyzed the functional consequences of this hetero-oligomeric unit on receptor trafficking.

We observed TRHR2 internalizes slower than TRHR1 in two different cell lines. In contrast, another study, which also compared the rates of internalization for the TRHR subtypes in COS-1 cells and more recently in HEK293 cells, reported a different rate for both TRHR1 and TRHR2 (14, 52). However, as the rate for TRHR1 internalization obtained by us is in agreement with that previously published by others (20, 23, 25, 26, 53, 54), we are unable to explain this discrepancy.

The majority of GPCRs, including TRHR1 and TRHR2, internalize via a clathrin and dynamin-dependent pathway involving -arrestin. Interestingly, the TRHR subtypes displayed a differential ability for -arrestin-promoted internalization, both -arrestin isoforms promoted TRHR1 internalization while -arrestin 2, and not -arrestin 1, promoted TRHR2 internalization.

These observations were strongly supported by experiments measuring the ability of the TRHRs to internalize in MEFs derived from -arrestin knockout mice (40). Internalization of TRHR1 was impaired when both -arrestins were absent. Importantly, internalization of TRHR2 was not affected in MEFs lacking -arrestin 1 but was impaired in MEFs lacking -arrestin 2 (either -arr2-KO or -arr1/2-KO). Similar results in MEF -arrestin KO cells were also obtained for the internalization of angiotensin 1A receptor, which utilizes both -arrestins, and the ₂-adrenergic receptor, which preferentially utilizes -arrestin 2 (40). A classification has been proposed where GPCRs that preferentially utilize -arrestin 2 for internalization are Class A receptors while GPCRs that utilize both -arrestins equally are Class B receptors (8, 9, 55). Therefore, TRHR2 is a Class A receptor, and TRHR1 is a Class B receptor.

The Class A and B classification is further defined by the ability of -arrestins to co-internalize with receptor, -arrestin 2 remains at the cell surface for Class A receptors, but internalizes with Class B receptors (56). In this study, we demonstrated that following prolonged activation of TRHR2 by TRH, -arrestin 2/GFP remained at the cell surface. In contrast, prolonged activation of TRHR1 by TRH, redistributed -arrestin 2/GFP into punctate vesicles. It has been shown that the receptor determinants for formation of these stable arrestin/receptor complexes are due to the presence of serine/threonine clusters in receptor carboxyl-terminal tails (C-tails) (56). For TRHR1, two such clusters exist in the C-tail, while for TRHR2 there are none, providing a possible explanation for determining whether co-internalization of receptor/-arrestin complexes occurs for each of the TRHR subtypes.

BRET, is a highly sensitive, quantitative approach to measure protein-protein interactions in living cells, in real time, and has been used by our group and others to detect GPCR interactions with -arrestin (30, 32). In this study, by utilizing BRET, we demonstrated an agonist-dependent interaction between TRHR2 and -arrestin 2, which is in agreement with our findings using more conventional techniques such as confocal microscopy. In addition, the highly sensitive BRET method provided evidence of a small, but consistent interaction between TRHR2 and -arrestin 1 which may play a role in TRHR2 desensitization, as shown for other Class A receptors (40).

There are a growing number of reports describing the existence of both functional receptor homo- and heterodimers, the latter of which can display pharmacological and functional properties distinct from those of the homodimer or oligomer. As TRHR subtypes are found to be co-expressed in various regions of the brain (14, 18), it was our aim to explore the potential of the TRHR subtypes to interact, and to examine any functional consequences. Similar to TRHR1 (30), TRHR2 formed constitutive homo-oligomers, which could be modulated upon addition of TRH. This increase in BRET ratio could represent either an increase in the number of oligomers and/or a change in the conformation of pre-existing complexes following ligand binding that results in a more favorable orientation of Rluc and EYFP moieties. BRET analysis also demonstrated constitutive receptor hetero-oligomerization between TRHR1 and TRHR2, which increased with TRH stimulation. An increasing number of reports of hetero-dimerization of other GPCR subtypes (1-5, 50, 57-60) have demonstrated that the hetero-oligomer functions differently to the homo-oligomer. However, while we observed no effect on TRH binding or signaling, the internalization kinetics in cells co-expressing TRHR1 and TRHR2 was significantly different from that in cells expressing either TRHR1, or TRHR2 alone. Likewise, heterodimers of the ₂-adrenergic receptor and kappa opioid receptors do not have significantly altered ligand binding or G-protein coupling properties of either receptor, but, the resulting heterodimer does display altered trafficking properties (61).

We wished to further understand the mechanisms underlying the altered trafficking of TRHR hetero-oligomers versus homo-oligomers. As the BRET demonstrated that TRHR subtypes differentially interacted with -arrestin 1 and 2, this provided us with a strategy for addressing a possible mechanism underlying the altered kinetics of TRHR hetero-oligomers trafficking. We found that the TRHR2/-arrestin 1 interaction as measured by BRET was enhanced by co-expression of TRHR1. This effect was not due to the ability of TRHR1 to efficiently interact with -arrestin 1, as a TRHR1 mutant (TRHR1 C335Stop) that does not interact with arrestins (26, 27, 31, 47), also promoted interaction of TRHR2 with -arrestin 1. Furthermore, cells co-expressing TRHR2 and TRHR1 C335Stop were able to rapidly translocate -arrestin 1/GFP. As neither receptor alone could induce a translocation of -arrestin 1, we suggest that formation of the TRHR1/TRHR2 hetero-oligomer resulted in a more favorable conformation that increased binding of -arrestin 1 to TRHR2, which may explain the change in internalization kinetics of the hetero-oligomeric unit. In addition, the TRHR1/2 hetero-oligomer also has Class A receptor characteristics in that -arrestin 1 remained at the cell membrane and did not redistribute into vesicles. To our knowledge this is the first observation that GPCR hetero-oligomers may have altered associations, compared with homo-oligomers, with intracellular regulatory proteins.

Overall, this study provides evidence that the TRHR subtypes are able to form hetero-oligomers in live cells and that this association may affect receptor trafficking by altering the association of TRHRs with -arrestins. A further understanding of the regulation and function of TRHR2 and the TRHR hetero-oligomeric complex may aid in explaining the diverse responses generated by a single ligand and assist in the design of therapeutic agents, which could selectively target the homo or heteromeric G-protein-coupled receptor subtypes.

	ACKNOWLEDGEMENTS

We thank Drs. K. Kroeger, M. Vrecl, and P. Tilbrook for critical reading of the article.

	FOOTNOTES

^* This work was supported by grants (to K. A. E.) from the National Health & Medical Research Council of Australia (Project Grant ID: 212065), the Raine Foundation, and the Keogh Institute for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: WAIMR, B Block, Sir Charles Gairdner Hospital, Hospital Ave., Nedlands, Perth, WA 6009, Australia. Tel.: 61-8-9346-1980; Fax: 61-8-9346-1818; E-mail: keidne@cyllene.uwa.edu.au.

Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M209340200

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; TRHR, thyrotropin-releasing hormone receptor; BRET, bioluminescence resonance energy transfer, MEF; mouse embryonic fibroblast, -arr-KO, -arrestin knockout; EYFP, enhanced yellow fluorescent protein; WT, wild type; GFP, green fluorescent protein; IP, inositol phosphate; Rluc, Renilla luciferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Chakera, R. M. Seeber, A. E. John, K. A. Eidne, and D. R. Greaves The Duffy Antigen/Receptor for Chemokines Exists in an Oligomeric Form in Living Cells and Functionally Antagonizes CCR5 Signaling through Hetero-Oligomerization Mol. Pharmacol., May 1, 2008; 73(5): 1362 - 1370. [Abstract] [Full Text] [PDF]

				G. J. Song, B. W. Jones, and P. M. Hinkle Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation PNAS, November 13, 2007; 104(46): 18303 - 18308. [Abstract] [Full Text] [PDF]

				A. Breit, K. Wolff, H. Kalwa, H. Jarry, T. Buch, and T. Gudermann The Natural Inverse Agonist Agouti-related Protein Induces Arrestin-mediated Endocytosis of Melanocortin-3 and -4 Receptors J. Biol. Chem., December 8, 2006; 281(49): 37447 - 37456. [Abstract] [Full Text] [PDF]

				S. Milasta, J. Pediani, S. Appelbe, S. Trim, M. Wyatt, P. Cox, M. Fidock, and G. Milligan Interactions between the Mas-Related Receptors MrgD and MrgE Alter Signalling and Trafficking of MrgD Mol. Pharmacol., February 1, 2006; 69(2): 479 - 491. [Abstract] [Full Text] [PDF]

				G. J. Song and P. M. Hinkle Regulated Dimerization of the Thyrotropin-Releasing Hormone Receptor Affects Receptor Trafficking But Not Signaling Mol. Endocrinol., November 1, 2005; 19(11): 2859 - 2870. [Abstract] [Full Text] [PDF]

				S. C. Prinster, C. Hague, and R. A. Hall Heterodimerization of G Protein-Coupled Receptors: Specificity and Functional Significance Pharmacol. Rev., September 1, 2005; 57(3): 289 - 298. [Abstract] [Full Text] [PDF]

				F. F. Hamdan, M. Audet, P. Garneau, J. Pelletier, and M. Bouvier High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based {beta}-Arrestin2 Recruitment Assay J Biomol Screen, August 1, 2005; 10(5): 463 - 475. [Abstract] [PDF]

				D. T. Dinh, H. Qian, R. Seeber, E. Lim, K. Pfleger, K. A. Eidne, and W. G. Thomas Helix I of {beta}-Arrestin Is Involved in Postendocytic Trafficking but Is Not Required for Membrane Translocation, Receptor Binding, and Internalization Mol. Pharmacol., February 1, 2005; 67(2): 375 - 382. [Abstract] [Full Text] [PDF]

				A. K. Scaffidi, N. Petrovic, Y. P. Moodley, M. Fogel-Petrovic, K. M. Kroeger, R. M. Seeber, K. A. Eidne, P. J. Thompson, and D. A. Knight {alpha}v{beta}3 Integrin Interacts with the Transforming Growth Factor {beta} (TGF{beta}) Type II Receptor to Potentiate the Proliferative Effects of TGF{beta}1 in Living Human Lung Fibroblasts J. Biol. Chem., September 3, 2004; 279(36): 37726 - 37733. [Abstract] [Full Text] [PDF]

				A. Breit, M. Lagace, and M. Bouvier Hetero-oligomerization between {beta}2- and {beta}3-Adrenergic Receptors Generates a {beta}-Adrenergic Signaling Unit with Distinct Functional Properties J. Biol. Chem., July 2, 2004; 279(27): 28756 - 28765. [Abstract] [Full Text] [PDF]

				L. B. Cook and P. M. Hinkle Fate of Internalized Thyrotropin-Releasing Hormone Receptors Monitored with a Timer Fusion Protein Endocrinology, July 1, 2004; 145(7): 3095 - 3100. [Abstract] [Full Text] [PDF]

				G. Milligan G Protein-Coupled Receptor Dimerization: Function and Ligand Pharmacology Mol. Pharmacol., July 1, 2004; 66(1): 1 - 7. [Abstract] [Full Text] [PDF]

				M. Pfeiffer, S. Kirscht, R. Stumm, T. Koch, D. Wu, M. Laugsch, H. Schroder, V. Hollt, and S. Schulz Heterodimerization of Substance P and {micro}-Opioid Receptors Regulates Receptor Trafficking and Resensitization J. Biol. Chem., December 19, 2003; 278(51): 51630 - 51637. [Abstract] [Full Text] [PDF]

				M. M. Berglund, D. A. Schober, M. A. Statnick, P. H. McDonald, and D. R. Gehlert The Use of Bioluminescence Resonance Energy Transfer 2 to Study Neuropeptide Y Receptor Agonist-Induced {beta}-Arrestin 2 Interaction J. Pharmacol. Exp. Ther., July 1, 2003; 306(1): 147 - 156. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/52/50422    most recent M209340200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hanyaloglu, A. C. Articles by Eidne, K. A. Search for Related Content PubMed PubMed Citation Articles by Hanyaloglu, A. C. Articles by Eidne, K. A. Social Bookmarking                      What's this?