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• W2007702516 abstract "A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity. A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity. The glycoprotein hormone receptors (GPHRs) 2The abbreviations used are: GPHR, glycoprotein hormone receptor; CG, choriogonadotropin; LHCGR, lutropin/CG receptor; FSHR, follicle-stimulating hormone receptor; TSH, thyroid-stimulating hormone; bTSH, bovine TSH; TSHR, TSH receptor; 7TMR, seven transmembrane receptor; TMH, transmembrane helix; ECD, extracellular domain; ECL, extracellular loop; ICL, intracellular loop; CAM, constitutively activating mutant; IP, inositol phosphate; WT, wild type; FACS, fluorescence-activated cell sorter. 2The abbreviations used are: GPHR, glycoprotein hormone receptor; CG, choriogonadotropin; LHCGR, lutropin/CG receptor; FSHR, follicle-stimulating hormone receptor; TSH, thyroid-stimulating hormone; bTSH, bovine TSH; TSHR, TSH receptor; 7TMR, seven transmembrane receptor; TMH, transmembrane helix; ECD, extracellular domain; ECL, extracellular loop; ICL, intracellular loop; CAM, constitutively activating mutant; IP, inositol phosphate; WT, wild type; FACS, fluorescence-activated cell sorter. LHCGR, FSHR, and TSHR constitute a subfamily of the 7TMRs. Members of the GPHRs are characterized by several common structural and functional features (1Szkudlinski M.W. Fremont V. Ronin C. Weintraub B.D. Physiol. Rev. 2002; 82: 473-502Crossref PubMed Scopus (326) Google Scholar, 2Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (700) Google Scholar, 3Ascoli M. Fanelli F. Segaloff D.L. Endocr. Rev. 2002; 23: 141-174Crossref PubMed Scopus (500) Google Scholar). Apart from the serpentine domain consisting of seven TMHs, three ECLs, three ICLs, and the intracellular C-terminal tail (see Fig. 1), the unique characteristic of GPHRs is a large N-terminal ECD, which is responsible for binding of the heterodimeric hormones (4Fan Q.R. Hendrickson W.A. Nature. 2005; 433: 269-277Crossref PubMed Scopus (460) Google Scholar). The GPHRs activate mainly Gαs and Gαq (2Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (700) Google Scholar, 5Laugwitz K.L. Allgeier A. Offermanns S. Spicher K. Van Sande J. Dumont J.E. Schultz G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 116-120Crossref PubMed Scopus (253) Google Scholar, 6Dufau M.L. Annu. Rev. Physiol. 1998; 60: 461-496Crossref PubMed Scopus (299) Google Scholar).TSHR is activated by TSH, CAMs (7Rodien P. Ho S.C. Vlaeminck V. Vassart G. Costagliola S. Ann. Endocrinol. (Paris). 2003; 64: 12-16PubMed Google Scholar, 8Kleinau G. Jäschke H. Neumann S. Lättig J. Paschke R. Krause G. J. Biol. Chem. 2004; 279: 51590-51600Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), mutants causing promiscuous hormone activity (9Costagliola S. Urizar E. Mendive F. Vassart G. Reproduction (Camb.). 2005; 130: 275-281Crossref PubMed Scopus (78) Google Scholar), antibodies (10Davies T.F. Ando T. Lin R.Y. Tomer Y. Latif R. J. Clin. Investig. 2005; 115: 1972-1983Crossref PubMed Scopus (200) Google Scholar), tryptic action (11Van Sande J. Massart C. Costagliola S. Allgeier A. Cetani F. Vassart G. Dumont J.E. Mol. Cell. Endocrinol. 1996; 119: 161-168Crossref PubMed Scopus (61) Google Scholar, 12Chen C.R. Chazenbalk G.D. McLachlan S.M. Rapoport B. Endocrinology. 2003; 144: 3821-3827Crossref PubMed Scopus (26) Google Scholar), small ligands (13Moore S. Jaeschke H. Kleinau G. Neumann S. Costanzi S. Jiang J.K. Childress J. Raaka B.M. Colson A. Paschke R. Krause G. Thomas C.J. Gershengorn M.C. J. Med. Chem. 2006; 49: 3888-3896Crossref PubMed Scopus (68) Google Scholar), and deletions of epitopes in the ECD (14Zhang M.L. Sugawa H. Kosugi S. Mori T. Biochem. Biophys. Res. Commun. 1995; 211: 205-210Crossref PubMed Scopus (48) Google Scholar) or the serpentine domain (15Duprez L. Hermans J. Van Sande J. Dumont J.E. Vassart G. Parma J. J. Clin. Endocrinol. Metab. 1997; 82: 306-308PubMed Google Scholar, 16Wonerow P. Schoneberg T. Schultz G. Gudermann T. Paschke R. J. Biol. Chem. 1998; 273: 7900-7905Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Here, for the first time, we provide a systematic evaluation of important functional properties of amino acids within the ECL2 of TSHR to gain insights into how ECL2 affects the signaling activity.Most pathogenic CAMs reported in the ECLs of TSHR are hydrophobic amino acids (ECL1, I486M,I486F (17Parma J. Van S e J. Swillens S. Tonacchera M. Dumont J. Vassart G. Mol. Endocrinol. 1995; 9: 725-733Crossref PubMed Google Scholar); ECL2, I568T,I568V (17Parma J. Van S e J. Swillens S. Tonacchera M. Dumont J. Vassart G. Mol. Endocrinol. 1995; 9: 725-733Crossref PubMed Google Scholar, 18Claus M. Maier J. Paschke R. Kujat C. Stumvoll M. Fuhrer D. Thyroid. 2005; 15: 1089-1094Crossref PubMed Scopus (33) Google Scholar); ECL3, N650Y, V656F (19Tonacchera M. Van S e J. Cetani F. Swillens S. Schvartz C. Winiszewski P. Portmann L. Dumont J.E. Vassart G. Parma J. J. Clin. Endocrinol. Metab. 1996; 81: 547-554PubMed Google Scholar, 20Fuhrer D. Holzapfel H.P. Wonerow P. Scherbaum W.A. Paschke R. J. Clin. Endocrinol. Metab. 1997; 82: 3885-3891PubMed Google Scholar)). Disruption of hydrophobic interactions caused by mutations at these positions may be responsible for structural rearrangements resulting in constitutive receptor activation. Because I568T,I568V in ECL2 are pathogenic CAMs (17Parma J. Van S e J. Swillens S. Tonacchera M. Dumont J. Vassart G. Mol. Endocrinol. 1995; 9: 725-733Crossref PubMed Google Scholar, 18Claus M. Maier J. Paschke R. Kujat C. Stumvoll M. Fuhrer D. Thyroid. 2005; 15: 1089-1094Crossref PubMed Scopus (33) Google Scholar), we assumed a tight hydrophobic interaction for Ile-568 in the partially active wild type basal conformation. A structural model of the serpentine domain of TSHR based on the rhodopsin structure (21Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4991) Google Scholar) orientates the ECL2 between the TMHs, where Ile-568 is located at the tip of ECL2 and is embedded in an environment of hydrophobic residues provided by TMH1, -2, -6, and -7. To identify hydrophobic residues at the TMHs that may interact with Ile-568 to constrain the basal conformation, we systematically tested amino acids at TMH1, -2, -6, and -7 that are located within a feasible interaction distance with Ile-568 suggested by the putative receptor structure. Indeed, the double mutant I568V/I640L suppressed constitutive cAMP signaling of I568V to wild type level, suggesting a structural and functional interplay between ECL2 and TMH6. Our findings provide new insights into structure-function relationships in GPHRs and highlight the importance of interactions between ECL2 and TMH6 for basal activity of TSHR.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis—The TSHR mutants were constructed by PCR mutagenesis using the human TSHR plasmid TSHR-pSVL as template as described previously (22Libert F. Lefort A. Gerard C. Parmentier M. Perret J. Ludgate M. Dumont J.E. Vassart G. Biochem. Biophys. Res. Commun. 1989; 165: 1250-1255Crossref PubMed Scopus (391) Google Scholar) PCR fragments were digested with BspTI and Eco91I (MBI Fermentas, Vilnius, Lithuania). The obtained fragments were used to replace the corresponding fragments in the WT TSHR-pSVL constructs. Mutated TSHR sequences were verified by dideoxy sequencing with dRhodamine terminator cycle sequencing chemistry (ABI Advanced Biotechnologies, Inc., Columbia, MD).Cell Culture and Transient Expression of Mutant TSHRs— COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Paisley, UK) at 37 °C in a humidified 5% CO2 incubator. Cells were transiently transfected in 12-well plates (1 × 105 cells/well) or 48-well plates (0.25 × 105 cells/well) with 1 μg of respective 0.25 μg of DNA/well using the GeneJammer® transfection reagent (Stratagene, Amsterdam, The Netherlands).FACS Analyses—The TSH receptor cell surface expression level was quantified on a FACS flow cytometer. Transfected cells were detached from the dishes with 1 mm EDTA and 1 mm EGTA in phosphate-buffered saline and transferred into Falcon 2054 tubes. Cells were washed once with phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% NaN3 and then incubated at 4 °C for 1 h with a 1:200 dilution of a mouse anti-human TSHR antibody (2C11, 10 mg/liter, Serotec Ltd., Oxford, UK) in the same buffer. Cells were washed twice and incubated at 4 °C for 1 h with a 1:200 dilution of fluorescein-conjugated F(ab′)2 rabbit anti-mouse IgG (Serotec). Before FACS analysis (FACscan, BD Biosciences) cells were washed twice and then fixed with 1% paraformaldehyde. Receptor expression was determined by the mean fluorescence intensity. The WT TSHR was set at 100%, and receptor expression of the mutants was calculated according to this. The percentage of signal positive cells corresponds to transfection efficiency, which was ∼60-70% of viable cells for each mutant.cAMP Accumulation Assay—For cAMP assays, cells were grown and transfected in 48-well plates. Forty-eight hours after transfection, cells were preincubated with serum-free Dulbecco's modified Eagle's medium containing 1 mm 3-isobutyl-1-methylxanthine (Sigma) for 20 min at 37 °C in a humidified 5% CO2 incubator. Subsequently, cells were stimulated with 100 milliunits/ml bTSH (Sigma) for 1 h. Reactions were terminated by aspiration of the medium. The cells were washed once with ice-cold phosphate-buffered saline and then lysed by the addition of 0.1 n HCl. Supernatants were collected and dried. cAMP content of the cell extracts was determined using the cAMP AlphaScreen™ assay (PerkinElmer Life Sciences, Zaventem, Belgium) according to the manufacturer's instructions.Stimulation of Inositol Phosphate Formation—Forty hours after transfection, cells were incubated with 2 μCi/ml myo[3H]inositol (18.6 Ci/mmol, Amersham Biosciences, Braunschweig, Germany) for 8 h. Thereafter cells were preincubated with serum-free Dulbecco's modified Eagle's medium without antibiotics containing 10 mm LiCl for 30 min. Stimulation by bTSH was performed in the same medium containing 100 milliunits/ml bTSH (Sigma) for 1 h. Intracellular inositol-phosphate (IP) levels were determined by anion exchange chromatography as described (23Berridge M.J. Biochem. J. 1983; 212: 849-858Crossref PubMed Scopus (763) Google Scholar). IP values are expressed as the percentage of radioactivity incorporated from [3H]inositol-phosphates (IP1-3) over the sum of radioactivity incorporated in IPs and phosphatidylinositols.Linear Regression Analysis of Constitutive Activity as a Function of TSHR Expression (Slopes)—The constitutive activity is expressed as basal cAMP formation as a function of receptor expression determined by 125I-bTSH binding. COS-7 cells were transiently transfected in 24-well plates (0.5 × 105 cells/well) with increasing concentrations of WT or mutant TSHR DNA (50, 100, 150, 200, 250, and 300 ng/well). For radioligand binding assays, cells were incubated in the presence of 160,000-180,000 cpm/ml of 125I-bTSH (BRAHMS Diagnostica, Berlin, Germany) supplemented with 5 milliunits/ml nonlabeled bTSH (Sigma). For cAMP assays, 48 h after transfection, cells were incubated with serum-free Dulbecco's modified Eagle's medium containing 1 mm 3-isobutyl-1-methylxanthine (Sigma) for 1 h. Cells were washed once with phosphate-buffered saline and then lysed using 0.1 n HCl. Supernatants were collected and dried. The cAMP levels were determined using the cAMP AlphaScreen Assay (PerkinElmer Life Sciences) according to the manufacturer's instructions. Basal cAMP formation as a function of receptor expression was analyzed according to Ballesteros et al. (24Ballesteros J.A. Jensen A.D. Liapakis G. Rasmussen S.G. Shi L. Gether U. Javitch J.A. J. Biol. Chem. 2001; 276: 29171-29177Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar) using the linear regression module of GraphPad Prism 2.01 for Windows.Molecular Modeling—The methods of molecular modeling procedures for TSHR serpentine domain have been previously described (25Jäschke H. Neumann S. Moore S. Thomas C. Colson A.O. Constanzi S. Kleinau G. Jiang J.K. Paschke R. Raaka B.M. Krause G. Gershengorn M. J. Biol. Chem. 2006; 281: 9841-9844Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The sheet-like fold of ECL2 and its general localization between the transmembrane helices were kept as in rhodopsin based on rhodopsin structure-consistent results for different accessibility of two CC chemokine receptor 5 (CCR5) antibodies, each specific for the two different β-strand epitopes of ECL2 of CCR5 (26Aarons E.J. Beddows S. Willingham T. Wu L. Koup R.A. Virology. 2001; 287: 382-390Crossref PubMed Scopus (42) Google Scholar, 27Dragic T. Trkola A. Lin S.W. Nagashima K.A. Kajumo F. Zhao L. Olson W.C. Wu L. Mackay C.R. Allaway G.P. Sakmar T.P. Moore J.P. Maddon P.J. J. Virol. 1998; 72: 279-285Crossref PubMed Google Scholar, 28Lee B. Sharron M. Blanpain C. Doranz B.J. Vakili J. Setoh P. Berg E. Liu G. Guy H.R. Durell S.R. Parmentier M. Chang C.N. Price K. Tsang M. Doms R.W. J. Biol. Chem. 1999; 274: 9617-9626Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar).RESULTSAlanine and Phenylalanine Mutations within ECL2All ECL2 residues (Fig. 1) were substituted by alanine (Table 1), and hydrophobic amino acids were also substituted with phenylalanine (supplemental Table 1). Wild type and mutated TSHRs were assessed after transient expression in COS-7 cells for cell surface expression, basal and TSH-stimulated cAMP, and IP accumulation. Cells transfected with a construct encoding the empty pSVL vector were used as controls.TABLE 1Functional characterization of alanine mutants at ECL2 COS-7 cells were transfected with the WT TSH receptor or described mutant TSH receptors. Functional assays were carried out as described under “Experimental Procedures.” Because of the basal activity of the WT TSHR, cAMP levels are expressed as relative to WT TSHR basal (set at 1). Increase in cAMP and IP levels was determined after stimulation with 100 milliunits/ml bovine TSH. The TSH receptor cell surface expression was quantified on a FACS flow cytometer. Data are given as mean ± S.D. of two independent experiments, each carried out in duplicate. The pSVL vector was used as a control.Transfected constructCell surface expression (FACS)CAMP accumulation (relative to WT basal)IP accumulationBasalStimulatedBasalStimulated% of WTWT100115.1 ± 0.11.8 ± 0.442.3 ± 4.5S561A58.7 ± 5.50.5 ± 0.0110.9 ± 0.41.8 ± 0.35.8 ± 0.7S562A50.3 ± 4.60.5 ± 0.0512.4 ± 0.61.9 ± 0.46.7 ± 1.2Y563A8.4 ± 0.90.6 ± 0.32.2 ± 0.51.9 ± 0.41.7 ± 0.5K565A74.6 ± 2.40.4 ± 0.12.1 ± 0.31.7 ± 0.22.5 ± 0.5V566A96.4 ± 8.21.4 ± 0.113.3 ± 0.61.9 ± 0.432.8 ± 4.0S567A108.7 ± 3.60.4 ± 0.111.6 ± 3.42.7 ± 0.427.6 ± 2.5I568A18.7 ± 2.52.2 ± 0.38.4 ± 0.81.7 ± 0.41.7 ± 0.3L570A46.9 ± 3.61.5 ± 0.19.4 ± 1.51.7 ± 0.47.1 ± 1.3M572A81.0 ± 8.30.2 ± 0.18.6 ± 0.71.8 ± 0.55.3 ± 0.6D573A63.6 ± 6.51.3 ± 0.111.1 ± 0.71.7 ± 0.428.7 ± 2.6T574A83.7 ± 8.81.7 ± 0.113.2 ± 1.31.9 ± 0.743.2 ± 2.2E575A82.8 ± 3.91.3 ± 0.310.3 ± 1.72.4 ± 0.537.2 ± 3.2pSVL0.4 ± 0.10.6 ± 0.012.0 ± 0.62.0 ± 0.6 Open table in a new tab We did not consider amino acids Cys-569 and Pro-571 of ECL2 in this mutagenesis approach since previous studies at both amino acids for the TSHR and the LHCGR (29Kosugi S. Ban T. Akamizu T. Kohn L.D. Biochem. Biophys. Res. Commun. 1992; 189: 1754-1762Crossref PubMed Scopus (45) Google Scholar, 30Ryu K. Lee H. Kim S. Beauchamp J. Tung C.S. Isaacs N.W. Ji I. Ji T.H. J. Biol. Chem. 1998; 273: 6285-6291Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) demonstrated that they are essential for correct folding of the receptors. For the FSHR, the threonine mutant of Pro-519 (TSHR: Pro-571) is known as an inactivating pathogenic mutant caused by intracellular trapping of the receptor (31Meduri G. Touraine P. Beau I. Lahuna O. Desroches A. Vacher-Lavenu M.C. Kuttenn F. Misrahi M. J. Clin. Endocrinol. Metab. 2003; 88: 3491-3498Crossref PubMed Scopus (148) Google Scholar). Amino acid Cys-569 is disulfide-linked to the conserved Cys-494 of TMH3 (Ballesteros and Weinstein number (32Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2413) Google Scholar) C3.25).Cell Surface Expression—FACS analyses revealed that the mutants show a cell surface expression in the range of 50-110% of WT TSHR with exception of mutants Y563A and I568A (Y563A 8.4%, I568A 18.7% of WT) (Table 1). We assume that the inactivity of the Y563A mutant is strongly related to its low number of receptors at the cell surface. Therefore, this mutant was not considered in further functional description.cAMP Accumulation—Alanine and phenylalanine mutants of Ile-568 are characterized by increased basal Gαs-mediated cAMP signaling when compared with WT (Table 1, supplemental Table 1). In contrast, mutants S561A, S562A, K565A, S567A, and M572A displayed decreased basal cAMP accumulation. For K565A, no basal cAMP signaling was observed (Table 1). TSH-mediated signaling is comparable with wild type or slightly decreased for all mutants (not under 50% when compared with maximum of WT) except mutant K565A with strongly impaired signaling activity.IP Accumulation—Basal inositol phosphate levels of all mutants were comparable with that of the WT TSHR (Table 1). Ligand-stimulated IP accumulation is markedly reduced by alanine mutants of Ser-561, Ser-562, Leu-565, Leu-570, and Met-572 (Table 1) and by V566F (supplemental Table 1).Potential Interaction Partners for Constitutively Activating Pathogenic Mutant I568VWe demonstrate that in addition to the known pathogenic CAMs I568T,I568V (17Parma J. Van S e J. Swillens S. Tonacchera M. Dumont J. Vassart G. Mol. Endocrinol. 1995; 9: 725-733Crossref PubMed Google Scholar, 18Claus M. Maier J. Paschke R. Kujat C. Stumvoll M. Fuhrer D. Thyroid. 2005; 15: 1089-1094Crossref PubMed Scopus (33) Google Scholar), mutants I568A,I568F (Table 1, supplemental Table 1) are CAMs. Therefore, we hypothesized a tight hydrophobic interaction for Ile-568 with its counterpart(s) to constrain the basal WT conformation since slight side chain alterations at Ile-568 lead to a release of these constraints. Moreover, we assumed that similar modifications at the counterpart side chains (shorter, longer, or bulkier when compared with WT TSHR) might also result in constitutive activation of the TSHR. The molecular model of the TSHR suggests that ECL2 is plugged nearly horizontally into the transmembrane domain on the extracellular side. Isoleucine 568 is located at the tip of ECL2 and is directly flanked by the conserved Cys-569, which is disulfide-bridged to Cys-494 at TMH3 and embedded between hydrophobic residues of TMH1, -2, -6, and -7. The side chain of Ile-568 points downwards in a hydrophobic cleft between the TMH1, -2, -6, and -7, and toward potential interaction partners Leu-417 (TMH1), Ile-470 (TMH2), Ile-640 (TMH6), and Val-664 (TMH7) (Fig. 2). Thus we tested these four potential hydrophobic interaction partners for Ile-568 constructing valine or alanine mutants (Table 2). Indeed, the I640V mutant in TMH6 with reduced side chain length was a CAM (basal activity 240%, WT TSHR set at 100%). Interestingly, further modifications of Ile-640 to methionine (elongated but flexible side chain), leucine (different branching), and phenylalanine (angled side chain) are not CAMs (Table 3). The I640L single mutant showed a decreased cAMP basal activity (basal activity ∼40%, WT TSHR set at 100%, Table 3). Now, we opted to test whether this phenotype affects mutant I568V. Combining the CAM I568V in ECL2 and I640L in TMH6 in the double mutant I568V/I640L resulted in restoration of the WT level of basal cAMP signaling (Table 3). The cAMP accumulation of the double mutant I568V/I640L after hormone stimulation was comparable with I568V (Table 3), whereas the decreased level of the IP accumulation was comparable with the single mutant I640L (Table 3).FIGURE 2Schematic representation of the localization of Ile-568 at ECL2 and potential interaction partners in the transmembrane region. The structural model of TSHR based on rhodopsin suggests a localization of Ile-568 at the tip of ECL2 embedded in a hydrophobic environment formed by the residues Leu-417 (TMH1), Ile-470 (TMH2), Ile-640 (TMH6), and Val-664 (TMH7).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Functional characterization of single mutants at TMH1, 2, 6 and 7 Potential hydrophobic interaction partners for I568 were constructed by substituting valine or alanine for the four potential interaction partners in TMH1, 2, 6, and 7. COS-7 cells were transfected with WT or mutant TSH receptors. Increase in cAMP and IP levels was determined after stimulation with 100 milliunits/ml bovine TSH. Data are given as mean ± S.D. of two independent experiments, each carried out in duplicate. The pSVL vector was used as a control.LocationTransfected constructCell surface expression (FACS)CAMP accumulation (relative to WT basal)IP accumulationBasalStimulatedBasalStimulated% of WTWT1001.0 ± 0.17.0 ± 0.92.6 ± 0.243.1 ± 3.1TMH1L417V98.1 ± 7.40.8 ± 0.15.6 ± 0.73.1 ± 0.28.5 ± 0.3TMH2I470V98.8 ± 7.50.9 ± 0.17.6 ± 1.63.1 ± 0.332.9 ± 2.0TMH6I640V95.9 ± 8.62.4 ± 0.310.1 ± 2.12.9 ± 0.245.5 ± 1.6TMH7V664A75.5 ± 8.51.1 ± 0.36.4 ± 1.12.7 ± 0.716.0 ± 0.7pSVL0.6 ± 0.10.6 ± 0.12.0 ± 0.02.1 ± 0.0 Open table in a new tab TABLE 3Functional characterization of single and double-mutants at ECL2 and TMH6 Increase in cAMP and IP levels was determined after stimulation with 100 milliunits/ml bovine TSH. Data are given as mean ± S.D. of two independent experiments, each carried out in duplicate. The pSVL vector was used as a control. ND = not determined.LocationTransfected constructCell surface expression (FACS)CAMP accumulation (relative to WT basal)IP accumulationBasalStimulatedBasalStimulated% of WTWT1001.0 ± 0.015.7 ± 0.82.9 ± 0.041.9 ± 0.9TMH6I640F74.2 ± 7.60.9 ± 0.18.7 ± 0.23.0 ± 0.27.7 ± 0.1TMH6I640M59.6 ± 2.61.2 ± 0.29.4 ± 1.64.2 ± 0.618.2 ± 0.1TMH6I640L92.5 ± 4.70.4 ± 0.17.8 ± 1.13.0 ± 0.112.7 ± 0.1TMH6I640V92.1 ± 2.02.9 ± 0.610.7 ± 1.5NDNDECL2I568V90.8 ± 2.12.9 ± 0.512.3 ± 0.62.7 ± 0.044.9 ± 0.0ECL2I568L101.0 ± 1.20.4 ± 0.0410.7 ± 1.6NDNDECL2/TMH6I568V,I640L76.1 ± 4.81.1 ± 0.011.2 ± 1.43.0 ± 0.118.7 ± 0.1ECL2/TMH6I568L,I640V98.5 ± 5.01.0 ± 0.19.8 ± 1.6NDNDpSVL0.3 ± 0.10.3 ± 0.13.5 ± 0.13.8 ± 0.1 Open table in a new tab Next, we tested the single mutants I568L, I640V and the double mutant I640V/I568L. Whereas I568V and all other known mutants at position 568 are CAMs (I568T,I568A,I568F (17Parma J. Van S e J. Swillens S. Tonacchera M. Dumont J. Vassart G. Mol. Endocrinol. 1995; 9: 725-733Crossref PubMed Google Scholar) (Table 1, supplemental Table 1)), mutant I568L is basally inactive (Table 3). This phenotype corresponds exactly to the phenotype of I640L. The combination of CAM I640V and mutant I568L with decreased basal cAMP activity in reciprocal double mutant I640V/I568L restored the basal cAMP activity of wild type. Linear regression analysis of TSHR mutants I568V and I640V confirmed increased basal cAMP activity (supplemental Fig. 1). The expression level for mutants I568L, I640V, and I568L/I640V is close to 100%, and the signaling capacity induced by TSH is about 70% of wild type.DISCUSSIONNumerous data for different 7TMRs have shown the importance of ECL2 for receptor signaling (33Sudo S. Kumagai J. Nishi S. Layfield S. Ferraro T. Bathgate R.A. Hsueh A.J. J. Biol. Chem. 2003; 278: 7855-7862Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 34Zoffmann S. Chollet A. Galzi J.L. Mol. Pharmacol. 2002; 62: 729-736Crossref PubMed Scopus (16) Google Scholar, 35Herold C.L. Qi A.D. Harden T.K. Nicholas R.A. J. Biol. Chem. 2004; 279: 11456-11464Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 36Seong J.Y. Wang L. Oh D.Y. Yun O. Maiti K. Li J.H. Soh J.M. Choi H.S. Kim K. Vaudry H. Kwon H.B. Endocrinology. 2003; 144: 454-466Crossref PubMed Scopus (41) Google Scholar). The ECL2 of the LHCGR has been reported to be involved in hormone binding (30Ryu K. Lee H. Kim S. Beauchamp J. Tung C.S. Isaacs N.W. Ji I. Ji T.H. J. Biol. Chem. 1998; 273: 6285-6291Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 37Couture L. Remy J.J. Rabesona H. Troalen F. Pajot-Augy E. Bozon V. Haertle T. Bidart J.M. Salesse R. Eur. J. Biochem. 1996; 241: 627-632Crossref PubMed Scopus (12) Google Scholar) and to be essential in signal transmission processes (38Li S. Liu X. Min L. Ascoli M. J. Biol. Chem. 2001; 276: 7968-7973Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). For the first time, the present study aimed to characterize systematically the amino acids within the ECL2 of TSHR by site-directed mutagenesis to provide deeper insights into the functional and structural interrelations during the processes of TSHR activation and intramolecular signal transduction.Lysine 565 in ECL2 Is a Key Player in the Intramolecular Signaling Processes of the TSHR—The alanine scan of ECL2 revealed K565A to be the most impaired mutant with respect to basal and hormone-induced activity (Table 1). We suggest two scenarios that might explain the low activity of K565A. (i) The cascade of constituents involved in the signal transmission process starting from the ligand-occupied ECD to intracellular effectors is interrupted by mutation of Lys-565 via breaking of hydrogen bonds or of electrostatic salt bridge interactions. (ii) Lys-565 is important for the formation of the active receptor state conformation by binding to a new interaction partner after hormone-induced signal initiation. The mutant is unable to stabilize the active receptor structurally and/or functionally.Mutant K565A at ECL2 is basally inactive and shows an impaired TSH-induced response. The recently published mutants K660D (TMH7), E409K, and D410K (e" @default.
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