Constitutive and Agonist-dependent Homo-oligomerization of the Thyrotropin-releasing Hormone Receptor

Abstract

The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or β2-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, β-arrestin, with TRHR and the absence of an interaction of β-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes. The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or β2-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, β-arrestin, with TRHR and the absence of an interaction of β-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes. Thyrotropin-releasing hormone (TRH)1 is involved in controlling the production of thyroid-stimulating hormone and prolactin from the anterior pituitary gland. TRH functions via binding to its receptor subtype that belongs to the large family of G-protein-coupled receptors (GPCRs), the first of which identified (1Zhao D. Yang J. Jones K.E. Gerald C. Suzuki Y. Hogan P.G. Chin W.W. Tashjian Jr., A.H. Endocrinology.. 1992; 130: 3529-3536Google Scholar, 2Straub R.E. Frech G.C. Joho R.H. Gershengorn M.C. 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Maya-Núñez G. Conn P.M. J. Biol. Chem... 2001; 276: 2153-2158Google Scholar) and heterodimerization between somatostatin receptor, somatostatin receptor 5, and the dopamine D2 receptor (29Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science.. 2000; 288: 154-157Google Scholar). Bioluminescence resonance energy transfer (BRET) represents a novel derivation of the FRET technique (37Xu Y. Piston D.W. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 151-156Google Scholar). This approach involves the transfer of energy resulting from the degradation of coelenterazine by Renilla luciferase (Rluc) to green fluorescent protein or a red-shifted variant, enhanced yellow fluorescent protein (EYFP), which in turn emits fluorescence. BRET is strictly dependent on the molecular proximity between the energy donor (Rluc) and acceptor (EYFP), making it ideal for studying protein-protein interactions. Furthermore, it has advantages over FRET in that it avoids the need for fluorescence excitation and thus possible cell damage and photobleaching (37Xu Y. Piston D.W. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 151-156Google Scholar). BRET was initially used to detect interactions between the cyanobacteria clock protein KaiB inEscherichia coli (37Xu Y. Piston D.W. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A... 1999; 96: 151-156Google Scholar). More recently, BRET has been used to demonstrate that the β2-adrenergic receptor exists as functional dimers in living cells and also the agonist-induced interaction between the receptor and adaptor protein β-arrestin 2 (38Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 3684-3689Google Scholar). Here we have taken advantage of this newly developed biophysical technique to investigate whether the TRHR could exist as oligomers in living mammalian cells. We have shown that in the unbound state, the TRHR exists as preformed homo-oligomeric complexes and that this interaction is modulated by agonist activation of the receptor. BRET was also used to examine the agonist-promoted interaction between TRHR and GnRHRs with the intracellular adaptor protein β-arrestin to demonstrate a direct real time interaction in intact cells. The TRHR/Rluc construct was generated by amplification of the rat TRHR coding sequence (39Sellar R.E. Taylor P.L. Lamb R.F. Zabavnik J. Anderson L. Eidne K.A. J. Mol. Endocrinol... 1993; 10: 199-206Google Scholar) without its stop codon using sense and antisense primers containingBamHI and NotI sites, respectively. The fragment was then cloned in frame into the pRluc vector constructed by insertion of the Rluc coding region into pcDNA3 (Invitrogen). Similarly, the TRHR/EYFP fusion construct was created by insertion of theBamHI/NotI fragment in frame into the EYFP vector constructed by insertion of the EYFP coding region into pcDNA3. The GnRHR/Rluc and GnRHR/EYFP constructs were generated by amplification of the rat GnRHR +1 stop codon mutant coding region (40Heding A. Vrecl M. Bogerd J. McGregor A. Sellar R. Taylor P.L. Eidne K.A. J. Biol. Chem... 1998; 273: 11472-11477Google Scholar) without its stop codon using sense and antisense primers containingEcoRV and NotI sites, respectively. This generated a 10-amino acid C-terminal extension to the GnRHR onto which the Rluc or EYFP coding sequence could be added. This GnRHREcoRV/NotI fragment was then cloned into the pcDNA3 vector containing either Rluc or EYFP as described above. The Rluc/EYFP fusion construct was generated by amplification of the Rluc coding region without its stop codon containing HindIII and EcoRV sites and insertion of thisHindIII/EcoRV fragment in frame into theHindIII/EcoRV sites of the EYFP vector. The rat GnRHR and rat GnRHR/TRHR carboxyl-terminal tail chimera (GnRHR/TRHR tail) have been previously described (40Heding A. Vrecl M. Bogerd J. McGregor A. Sellar R. Taylor P.L. Eidne K.A. J. Biol. Chem... 1998; 273: 11472-11477Google Scholar). The cDNA for human protease-activated receptor 1 was provided by L. Brass (University of Pennsylvania, Philadelphia, PA). The dynamin dominant negative mutant (K44A) construct was provided by S. Schmid (University of California, San Francisco, CA). The β2-adrenergic/Rluc (β2-AR/Rluc) and β2-adrenergic/EYFP (β2-AR/EYFP) fusion constructs were provided by M. Bouvier (University of Montreal) and were previously described (38Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 3684-3689Google Scholar). HEK 293 and COS 1 cells (ATCC) maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, glutamine (0.3 mg/ml), and penicillin/streptomycin (100 units/ml) (Life Technologies, Inc.) at 37 °C in 5% CO2 were seeded at a density of 5 × 105 or 1 × 106 cells/ml per 60- or 100-mm dish, respectively. Transient transfections were performed the following day with 5 or 10 μg of total DNA using Superfect (Qiagen), and cells were used 48 h post-transfection. Iodinated [d-Trp6,Pro9,N-Et]GnRH (Sigma) was prepared using the lactoperoxidase method and purified by chromatography on a Sephadex G-25 column in 0.01 m acetic acid/0.1% bovine serum albumin. The specific activity was 42 μCi/μg and was calculated as described previously (41Bramley T.A. McPhie C.A. Menzies G.S. Placenta.. 1992; 13: 555-581Google Scholar). Transfected HEK 293 cells were plated onto poly-l-lysine-coated eight-well chamber slides 24 h after transfection. Treatments were carried out 48 h post-transfection, and then cells were fixed in 4% paraformaldehyde, mounted in Fluoroguard (Bio-Rad), and sealed with a coverslip. Cells were excited with a 488-nm laser light and examined using a Bio-Rad confocal laser microscope under an oil immersion × 60 objective with light filtered in the green channel from 500 to 550 nm. Dose displacement receptor binding assays were performed on COS 1 cells transiently transfected with untagged and tagged TRHR or GnRHR 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) with125I-labeled [d-Trp6,Pro9,N-Et]GnRH or [3H][Me-His2]TRH (PerkinElmer Life Sciences) with or without unlabeled agonist. The concentration of unlabeled agonist used ranged from 10−6 to 10−11m. The cells were then washed and solubilized in 0.2 m NaOH, 1% SDS, and the radioactivity was counted, with receptor binding expressed as a percentage of maximum specific binding. Receptor internalization assays were performed in HEK 293 cells as previously described (42Vrecl M. Anderson L. Hanyaloglu A. McGregor A.M. Groarke A.D. Milligan G. Taylor P.L. Eidne K.A. Mol. Endocrinol... 1998; 12: 1818-1829Google Scholar). Briefly, transiently transfected cells in 24-well plates were incubated with 125I-labeled [d-Trp6,Pro9,N-Et]GnRH or [3H][Me-His2]TRH at 37 °C for 5–120 min. Surface bound radioactivity was then removed by washing with acid solution for 12 min. Internalized radioactivity was determined after solubilizing cells in 0.2 m NaOH, 1% SDS. Nonspecific binding for each time point was determined under the same conditions in the presence of 1 μm unlabeled GnRH agonist (leuprolide) or TRH. After subtraction of nonspecific radioactivity, internalized radioactivity was expressed as a percentage of the total binding. All time points were performed in duplicate in at least three separate experiments. Forty-eight hours post-transfection, COS 1 cells were detached with phosphate-buffered saline plus 0.05% trypsin and washed twice in phosphate-buffered saline. Approximately 50,000 cells/well were distributed in a 96-well plate and incubated in the presence or absence of TRH (Sigma), GnRH agonist, leuprolide (Abbott Australasia), or GnRH antagonist, Antide (Sigma) (all 10−6m final concentration) for the specified time period at 37 °C. The coelenterazine (h form) (Molecular Probes, Inc., Eugene, OR) 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) which allows sequential integration of the signals detected in the 440–500 and 510–590 nm windows. Data are represented as a normalized BRET ratio, which is defined as the BRET ratio for the co-expression of the Rluc and EYFP constructs normalized against the BRET ratio for the Rluc expression construct alone (38Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A... 2000; 97: 3684-3689Google Scholar). The BRET ratio is defined as ((emission at 510–590 nm) − (emission at 440–500 nm) ×cf)/(emission at 440–500 nm), where cfcorresponds to (emission at 510–590 nm/emission at 440–500 nm) for the Rluc construct expressed alone in the same experiment. TRHR fusion proteins with either Rluc or EYFP added to the C terminus were validated to ensure they displayed functional characteristics similar to untagged, wild-type receptors. Confocal microscopy was used to demonstrate that the TRHR/EYFP fusion protein was expressed on the plasma membrane (Fig.1 A, I). binding and internalization assays were used as an to receptor expression at the cell as as receptor function with TRHR/Rluc and TRHR/EYFP were shown to at the cell and displacement binding assays similar with wild-type I). Furthermore, the of TRHR/Rluc fusion receptor internalization was not in with untagged receptor in to TRH (Fig.1 The TRHR/EYFP fusion receptor, however, 10% internalization with wild-type for wild-type and BRET fusion cell binding assays were carried out in COS 1 cells either wild-type or receptor fusion proteins with of unlabeled agonist to TRHR and GnRHR displacement binding assays were performed using [3H][Me-His2]TRH and respectively. shown are the of three experiments. in a new cell binding assays were carried out in COS 1 cells either wild-type or receptor fusion proteins with of unlabeled agonist to TRHR and GnRHR displacement binding assays were performed using [3H][Me-His2]TRH and respectively. shown are the of three experiments. In order to of the interaction between TRHRs, EYFP and Rluc fusion constructs of another GPCR, the were generated and validated as described above. stop codon mutant a GnRHR with a 10-amino acid C-terminal described (40Heding A. Vrecl M. Bogerd J. McGregor A. Sellar R. Taylor P.L. Eidne K.A. J. Biol. Chem... 1998; 273: 11472-11477Google Scholar) was to the the GnRHR/EYFP was expressed on the plasma as by confocal fluorescence was suggesting the presence of an intracellular of receptors (Fig.1 A, Receptor binding assays and internalization assays in cells each of the GnRHR fusion constructs the expression of GnRHR/EYFP and GnRHR/Rluc at the cell The GnRHR has previously been shown to at a with the TRHR in HEK 293 and COS cells (42Vrecl M. Anderson L. Hanyaloglu A. McGregor A.M. Groarke A.D. Milligan G. Taylor P.L. Eidne K.A. Mol. Endocrinol... 1998; 12: 1818-1829Google Scholar). the binding the internalization 1 characteristics of either of the and forms of the GnRHR were with wild-type receptor. whether constitutive interaction between TRHRs in living cells, BRET was in COS 1 cells either TRHR/Rluc and pcDNA3 vector or TRHR/Rluc and TRHR/EYFP fusion The ratio of concentration used for was of fluorescent acceptor expression over donor is thought to energy and using a of of TRHR/Rluc to TRHR/EYFP DNA that the ratio in an BRET ratio not the of the luciferase a signal was in the and in cells TRHR/Rluc and vector. the BRET the ratio of light in the over that in the was determined as described under Cells TRHR/Rluc and TRHR/EYFP an increase in the amount of light in the to the of and thus an increase in the BRET ratio of to the BRET ratio for TRHR/Rluc only out the that BRET was between transfected cells that were either of the TRHR fusion not in the same either TRHR/Rluc or TRHR/EYFP were BRET from cells not were with cells either TRHR/Rluc alone or tagged of TRHR/Rluc and TRHR/EYFP in the same cells was for BRET to thus evidence that an interaction was in the same that the evidence the existence of TRHR we to investigate whether agonist activation the or of TRHR agonist of COS 1 cells TRHR/Rluc and an increase in the BRET signal was in with increase in the BRET signal was either or GnRH agonist was The effect of receptor activation on TRHR/Rluc and TRHR/EYFP energy transfer with time of to agonist, a maximum following a In a dose-dependent increase in agonist the BRET ratio with an of in a similar to that for ligand binding I). examine the between receptor expression and BRET of TRHR/Rluc and TRHR/EYFP at a ratio were transfected into COS 1 cells. BRET was shown to increase with of a at the concentration of μg of TRHR/Rluc with μg of TRHR/EYFP of receptor expression by binding with of TRH receptor expression on BRET COS 1 cells were with of DNA for TRHR/Rluc and as The BRET ratio was immediately following the of The same of transfected cells were also used in cell binding assays are expressed as the percentage of maximum ratio for each were carried out at least three and shown are The BRET between the TRHR/Rluc and TRHR/EYFP did not from nonspecific interaction between the Rluc and EYFP of the fusion increase in the signal was detected Rluc and EYFP were co-expressed as proteins, with Rluc expressed alone not This is in to the BRET signal generated by a fusion construct of Rluc to EYFP used as a which an BRET ratio over that of Rluc alone not The increase in the of the BRET ratio upon co-expression of Rluc and EYFP or is to a of nonspecific of Rluc and EYFP as a of and fusion constructs were co-expressed with the TRHR fusion proteins, in order to for the of the TRHR/Rluc and TRHR/EYFP interaction and to out the that it is not to receptor at the of the TRHR/Rluc and GnRHR/EYFP did not to an energy transfer in either cells or in cells with either TRH or GnRH agonist alone or similar was for cells the of either the TRHR/EYFP with the GnRHR/Rluc or TRHR/Rluc with the 5 The of BRET in COS 1 cells the GnRHR/Rluc and GnRHR/EYFP with either GnRHR/Rluc with GnRHR/EYFP with or GnRHR/Rluc alone that the GnRHR may not form oligomers in the absence of However, following the of GnRH agonist, an increase in the BRET ratio was 5 that an interaction between GnRHR/Rluc and GnRHR/EYFP occur. The of a GnRH effect on BRET not demonstrate the of the TRHR untagged TRHR was transfected into COS 1 cells in to TRHR/Rluc and of untagged TRHR was to the BRET signal generated between the TRHR/Rluc and TRHR/EYFP 5 In expression of other untagged GPCRs the GnRHR/TRHR chimera and protease-activated receptor did not the BRET signal between the two tagged TRH receptors 5 receptor expression was by ligand binding assays not in BRET was the GnRHR/TRHR tail chimera was that the TRHR C-terminal tail is not involved in the formation of this receptor. This that the BRET between TRHR/Rluc and TRHR/EYFP is to a specific interaction between at least two TRHR and evidence for the existence of preformed or constitutive receptor Agonist stimulation is known to clustering and internalization of the TRHR R. Hinkle P.M. J. Biol. Chem... 1999; 274: 15745-15750Google Scholar, M. Anderson L. Hanyaloglu A. McGregor A.M. Groarke A.D. Milligan G. Taylor P.L. Eidne K.A. Mol. 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