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Third Medical Department, University of Leipzig (S.N., M.C., R.P.), D-04103 Leipzig, Germany; and Institute for Molecular Pharmacology (G.K.), D-13125 Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Ralf Paschke, Third Medical Department, University of Leipzig, Ph. Rosenthal Strasse 27, 04103 Leipzig, Germany. E-mail: ralf.paschke{at}medizin.uni-leipzig.
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Introduction |
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ICL2 seems to be important for the selectivity of receptor/G protein interactions and the efficiency of G protein activation (3). Investigation of ICL2 in the m5 muscarinic receptor by a random mutagenesis approach has shown that ICL2 contains a group of residues necessary for holding the receptor in an inactive conformation and another group necessary for G protein coupling. Therefore, it was hypothesized that ICL2 could act as a switch that enables G protein coupling (6). Moreover, in ICL2 of the m3 muscarinic receptor, four specific amino acids (aa) were identified as important for enabling the receptor to couple selectively to the Gq/11 protein (7). Recent findings for metabotropic receptors, which belong to class 3 of 7TMRs, underline the importance of ICL2 by demonstrating that the central portion of ICL2 is responsible for the selective recognition of the C-terminal end of the G
-subunit (8).
The TSH receptor (TSHR) together with the LH and FSH receptors belong to the glycoprotein hormone receptor family. TSHR activation by several activating point and deletion mutants within ICL3 and the lower portion of TM6 supports a direct G protein activation by this region (9, 10). Interestingly, naturally occurring, activating TSHR mutations, which are one of the molecular causes of autosomal dominant, nonautoimmmune hyperthyroidism and toxic thyroid nodules (9, 11) (OMIM 603372), were predominantly localized to TM domains, extracellular loops, or ICL3 and rarely in the first or second ICL (12). For this reason many studies have focused on residues in ICL3, but only two studies have focused on ICL2 of the TSHR (13, 14). The first study concluded that the carboxyl-terminal domain of ICL2 (aa 528–537) is important for activation of Gs-mediated cAMP production (13). TSHR mutants created by substituting sequences from the 1- or ß2-adrenergic receptor demonstrated that the middle portion of ICL2 (residues 525–527) of the TSHR seems to be important for agonist-induced Gs interaction, whereas residues 528–532 were determined to be more critical for agonist-induced Gq activation (14). Mutagenesis studies of LH and FSH receptors have also provided evidence for an important functional role of several aa residues within ICL2 (15, 16). However, only single selected residues in ICL2 were substituted in these studies.
Less is known about the specific function of single residues of ICL2 and potential interaction with ICL3 in dual G protein activation by glycoprotein hormone receptors. Therefore, the first aim of our study was to identify and characterize specific intracellular aa of the TSHR involved in G protein activation and selectivity. In contrast to both former studies (13, 14), in which several adjacent aa within ICL2 of the TSHR were replaced simultaneously, systematic single site mutagenesis of the entire ICL2 was carried out. The second aim was to identify potential intramolecular interactions between ICL2 and ICL3 that might facilitate G protein activation. Deletions of four or five consecutive residues and multiple alanine mutations of the same residues (Fig. 1
) localized the ICL2 region important for TSHR activation to residues 523–530. Our data reveal that M527 and, to a lesser extent, F525, R528, L529, and D530 are residues with selective influence on Gq activation. Double mutants between ICL2 and ICL3 suggest interaction between these loops in the vicinity of F525 and T607 during TSHR activation. Moreover, our data exclude an interaction of M527 with ICL3, supporting the idea of direct selective interaction of M527 with the Gq-subunit, which has already been suggested for other 7TMRs (17, 18).
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Materials and Methods |
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Cell culture and transfection
COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies, Inc., Paisley, UK) at 37 C in a humidified 7% CO2 incubator. Cells were transiently transfected in 12-well plates (1 x 105 cells/well) with 1 µg DNA/well using the FuGene 6 reagent (Roche, Basel, Switzerland).
Radioligand binding assay
Competitive binding studies were performed as previously described (10). Data were analyzed assuming a one-site binding model using the fitting module of SigmaPlot 2.0 for Windows (21).
FACS analysis
Forty-eight hours after transfection, nonpermeabilized cells were detached from the dishes using 1 mM EDTA and 1 mM EGTA in PBS and transferred in Falcon 2052 tubes. Before incubation with the primary antibody, cells were washed once with PBS containing 0.1% BSA and 0.1% NaN3. Afterward, cells were incubated with a mouse antihuman TSHR antibody (2C11, Serotec, 10 µg/ml) in the same buffer. Tubes were washed and incubated for 1 h on ice in the dark with fluorescein-conjugated F(ab')2 rabbit antimouse IgG (Serotec, Oxford, UK; dilution, 1:1000). Before FACS analysis (FACScan, BD Biosciences), cells were washed twice and fixed with 1% paraformaldehyde. Receptor expression was determined by the fluorescence intensity, whereas the percentage of signal-positive cells corresponds to the transfection efficiency.
cAMP accumulation assay
Measurement of cAMP accumulation was performed 48 h after transfection, as previously described (10).
Stimulation of inositol phosphate (IP) formation
Transfected COS-7 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 DMEM, without antibiotics, containing 10 mM LiCl for 30 min. Stimulation with TSH was performed with the same medium supplemented with 100 mU/ml TSH for 1 h. Basal and TSH-induced increases in intracellular IP levels were determined by anion exchange chromatography as previously described (22). IP values are expressed as the percentage of radioactivity incorporated from [3H]IP (IP1–3) over the sum of radioactivity incorporated in IPs and phosphatidylinositols.
Molecular modeling
The packing of the seven-helix backbone of the rhodopsin structure (23) was used as a template for the TM regions of the TSHR. The TSHR structure model was computed with special emphasis on the TM and intracellular portions, without the large amino-terminal ectodomain, but including the extra- and intracellular loops. The starting conformation of ICL1 and ICL2, the first portion of the C-terminal tail formed as helix 8, and the overall fold of the second extracellular loop were also adopted from the rhodopsin structure. For the remaining parts of the ICLs as well as for the extracellular loops, fragments of four to seven residues were selected and tested against the three-dimensional protein database. Only fragments occurring more than once with a similar backbone conformation in the database were used for assembling the loops. All model components were assembled with the biopolymer module of the SYBYL program package (TRIPOS, Inc., St Louis, MO). Simulated annealing runs were performed by heating up to 1,000 K 100 times, equilibrating for 2,000 psec, and cooling to 0 K during 10,000 psec. The C- atoms of the TM helices were restrained in place by a punctual harmonic potential. For ICL2 and ICL3, two main conformational clusters were sampled. For one of the two clusters, molecular dynamic simulations were performed at 300 K for 1 nsec, where only the helix stability was maintained by restraints for hydrogen bonds of the TM backbones. For all energy and dynamics calculations, the AMBER 5.0 force field (24) was used. The geometrical quality of the two resulting models, A and B, was controlled using the program PROCHECK (25).
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Results |
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1 from aa positions 522–525, 2 from aa positions 526–530, and 3 from aa positions 531–534 (Fig. 1
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Although characterized by a slightly increased cell surface expression compared with the wt TSHR, all three deletion mutants were inactive for TSH-stimulated IP production and strongly impaired for cAMP production (Table 1). All three deletion constructs had decreased basal cAMP activity compared with wt and TSH-stimulated activity that was substantially lower than wt. These findings suggest that G protein coupling was impaired in each deletion construct.
Multiple alanine substitutions (ASU)
To clarify whether the impaired G protein coupling is caused by deletion-related conformational changes or by specific aa residues within ICL2, ASU were introduced in place of the deletions. The ASUs span the following regions: ASU1 from aa 522–525, ASU2 from aa 526–530, and ASU3 from aa 531–534, corresponding to 1–3 (Fig. 1). The ASUs showed gradually decreased impairment of TSH-stimulated receptor activity for the cAMP pathway as they progressed toward the region of ICL2 nearest TM4 (Fig. 2 and Table 1). In contrast, the Gq-mediated IP pathway was not activated by TSH in any of the ASUs (Fig. 2 and Table 1). Because the ASU3 mutant showed slightly decreased cell surface expression compared with the wt TSHR, the TSH-stimulated cAMP accumulation for this mutant may be underestimated. These findings indicate that the N-terminal and middle portions of ICL2 seem to be more important for Gs activation than the region nearest TM4 and, moreover, reveal a critical importance of ICL2 for Gq activation.
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and Table 1). These substitutions within the region from position R531 to R534 revealed no influence on Gs protein coupling (Table 1), confirming the finding of increased cAMP accumulation for ASU3 compared with ASU1 and ASU2. A lower IP accumulation for R531A after stimulation with TSH and a 2.5-fold higher EC50 for both R531A and K532A (Table 1) may partially account for the inactivation of IP production in the ASU3 mutant.
Single ASUs within the region from I523 to D530 revealed stronger differences between Gs- and Gq-dependent signaling, which could be pinpointed to specific aa. TSHR mutant I523A exhibited a weak TSH-induced cAMP accumulation, a 5.6-fold higher EC50 than wt TSHR, and no IP stimulation (Table 1 and Fig. 3, A and B). COS-7 cells transfected with I523A showed cell surface expression of 70% relative to wt TSHR, which does not explain the strongly decreased cAMP and inactive IP production of this mutant. R534A, for example, showed a comparable cell surface expression without significant impairment of the cAMP and IP signals (Table 1). ASUs within the region of 1 (aa 522–525), in which only one wt aa residue was preserved (AIAA, AATA, and AAAF; Fig. 1) confirm the importance of I523, because the AIAA substitution in which I523 was intact showed the best Gs coupling of these three mutants (Table 1).
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and Fig. 3C). After maximal stimulation with TSH, this mutant showed a cAMP accumulation 45% that of wt TSHR, with an EC50 that was 2.5-fold higher than the wt value. However, no TSH-stimulated IP production was measured (Table 1 and Fig. 3, C and D). Thus, M525A is inactive for the Gq-coupled pathway and loses the dual G protein signaling characteristic of the TSHR.
A selective impairment of TSH-induced phosphoinositide hydrolysis was also observed for the mutants F525A, R528A, L529A, and D530A. cAMP production was near wt TSHR levels in these mutants, with only slightly lower maximum TSH-induced cAMP production and modest increases in EC50 values (Table 1 and Fig. 3C). In contrast, IP production of these mutants was between 19–42% of the wt TSHR, and the EC50 values increased 2.4- to 3.3-fold (Fig. 3D). Mutant T524A, which does not show reduced maximum cAMP or IP production, did exhibit a 2.7-fold increased EC50 for Gq activation (Table 1 and Fig. 3, A and B). Because cell surface expression for T524A was only 75% compared with that of wt TSHR, the observed reduced cell surface expression of R528A and D530A (Table 1) most likely does not account for the reduced TSH-stimulated IP production of those mutants. Taken together, these data demonstrate that the most important residues for Gs/Gq coupling and, in particular, for selective loss or impairment of Gq coupling are localized in the N-terminal and middle portions of ICL2 at position I523 and within the region from F525 to D530.
Molecular modeling
To study the ICLs, TSHR lacking the large N-terminal ectodomain was modeled based on the rhodopsin structure template. However, the ICL2 conformation of rhodopsin could only be used as a starting point, because the ICL2 of the TSHR is one residue longer and lacks sequence similarity in its hydrophobic residues compared with rhodopsin. Concerning ICL2 and ICL3, simulated annealing runs provided two different clusters of conformations. The conformational stability of each was confirmed using molecular dynamic simulations of one representative. The main difference in the resulting two models is the orientations of F525 and M527 (Fig. 5). In model A, F525 and M527 are constituents of a large hydrophobic core stabilizing the fold of the TM3/ICL2 junction, whereas in model B, F525 is interacting with the ICL3 in the region of T607. Residue M527 points to the cytosol toward ICL3. In our simulated annealing studies the M527 side chain was observed to be rather flexible, moving between the hydrophobic ICL2 core, an intracellular orientation, and an orientation toward ICL3 (Y613). Common to both models is the conformation of the TM3/ICL2 junction, which is dominated by a hydrophobic core formed by W520 and Y521, which are still members of TM3, and the residues I529 and I533, which only escort this cluster. The residue I523 is the last hydrophobic residue of TM3 pointing to a hydrophobic patch between TM5 and TM6. The C-terminal portion of ICL2, comprised of residues 528–534, forms an extended conformation toward TM4.
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). This inactive phenotype of M527K for both signaling cascades was maintained in the double mutants with T607E and Y613E (Table 2), suggesting that M527 (ICL2) does not interact with these residues in ICL3. In contrast to position M527, the phenotypes of F525A and F525K were comparable for Gs and Gq signaling (Tables 1 and 2). The double mutants, F525K/Y613E and F525K/T607E, showed functional properties similar to those of the wt TSHR for cAMP production (Table 2 and Fig. 4, A and B). However, strong differences between both double mutants were observed regarding Gq-mediated signaling. The double mutant F525K/T607E showed a rescue of the Gq-coupled pathway by a 24% higher maximal IP accumulation compared with F525K alone (Table 2 and Fig. 4C). The EC50 shift for this double mutant was comparable to that with F525K alone. In contrast, F525K/Y613E exhibited a 14% lower IP production and a 2.2-fold higher EC50 compared with F525K alone (Fig. 4D). These data suggest that ICL2 interacts with ICL3 in the vicinity of F527 and T607 and provides support for the tertiary fold proposed in model B above (Fig. 5B).
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Discussion |
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Identification of key residues in ICL2 for the dual G protein activation by TSHR
All three deletions within ICL2 are expressed comparable to the wt TSHR, but Gs/Gq protein coupling was abolished or strongly diminished. It is conceivable that these deletions result in TSHR conformations that allow the receptors to reach the cell surface, but do not allow productive contact with G proteins. To investigate the cause of the disturbance in intermolecular signal transduction in more detail and to gain information at the level of single aa, we introduced alanine residues within the regions of the deletion mutants. The stepwise increase in reconstitution of cAMP activity from ASU1 to ASU3 toward TM4 was a decisive indication that specific residues in the N-terminal half of ICL2 were probably involved in Gs coupling. Moreover, the complete inhibition of Gq-mediated IP production by ASU1–3 indicated a functional impact of ICL2 on this pathway. The substitution of single aa with alanine confirmed these observations, showing that the region of F525 to D530 has a slight effect on Gs signaling, but plays a more important role in Gq signaling.
The strongest change regarding selective loss of G protein activation was observed with M527A. Indeed, the efficiency of Gs coupling was affected only slightly, whereas this mutant lost the ability to stimulate Gq-dependent phospholipase C activation despite a normal cell surface expression. Interestingly, Y601H in TM5 of TSHR near the border with ICL3 is also a determinant for Gq coupling, because this mutant does not signal via Gq, but cAMP formation remains unaltered (26). Similarly, Y601N (27) confirms the functional role of Y601 in the activity and dual signaling capability of the TSHR. Taken together, these and our data are consistent with the observation that residues involved in G protein coupling and selectivity are primarily localized at the TM/cytoplasmic borders between TM3/ICL2, TM5/ICL3, and ICL3/TM6 (1).
The extension of this selective effect on Gq activation up- and downstream of M527 (F525A and R528A to D530A) fits well with the idea that receptor domains, rather than single aa, participate in forming a G protein binding pocket. Our findings are in part consistent with the data reported by Kosugi et al. (14) showing that the most important determinant for agonist-increased cAMP production is the region around residues 525–527 of ICL2 of the TSHR. However, they observed a total loss of TSH-induced cAMP response and a lesser loss in phosphoinositide signaling for a mutant in which residues 525–527 of the TSHR were substituted with a comparable ß2-adrenergic receptor sequence. Furthermore, single ASUs at positions 528–534 do not or only slightly affect Gs activation, which seems contradictory to the findings by Chazenbalk et al. (13). One explanation for these differences might be that simultaneous substitution of three or more residues has a stronger effect than single substitutions, which we also observed with our deletions and ASU mutants.
Different tasks of hydrophobic residues F525 and M527
F525: experimental indication for an interaction between ICL2 with ICL3.
The main differences in the proposed models are the orientations of F525 and M527. In the ICL2 stabilization model (A), F525 participates in a large hydrophobic core, stabilizing the fold of the TM3/ICL2 junction, whereas in the ICL2-ICL3 interaction model (B), F525 in ICL2 points toward ICL3 to residue T607 and in the vicinity of Y613. Drastic alteration of hydrophobic properties at position F525 by alanine and lysine has only a weak impact on TSH-induced cAMP formation, but, in contrast, the stimulated IP accumulation is clearly decreased. Interestingly, only the double mutant F525K/T607E achieved a partial functional rescue of stimulated IP production. In contrast, with the double mutant F525K/Y613E, TSH-induced IP formation was further decreased. Despite increased cell surface expression, Gs- and Gq-dependent signaling was strongly inhibited by M527K. Double mutants between M527 at ICL2 and Y613 and T607 at ICL3 did not lead to a functional rescue for cAMP or IP production. Taken together, a cooperative effect between ICL2 and ICL3 in the vicinity of F525 and T607 is indicated, supporting the tertiary fold proposed in model B.
M527 and its vicinity from F525 to D530 play pivotal roles in Gq-dependent signaling
Our findings suggest that the M527 side-chain is essential for Gq-mediated signaling and confirm previous findings with other 7TMRs that a methionine or another hydrophobic residue at the position of M527 could play a key role in selective signaling toward Gq (26, 28). M145 in the V2 vasopressin receptor, which is located at a position identical to M527 in the TSHR, was identified as a residue with pronounced effects on receptor/G protein coupling selectivity (18). The presence of a relatively large hydrophobic aa side-chain such as leucine or tryptophan at position 145 favored Gq signaling and suggests a recognition of the C terminus of Gq by this residue (18). This fits with our observation that alterations of the hydrophobic properties at M527 in the TSHR have the strongest selective effect on Gq protein activation. The corresponding position to M527 in the GnRH receptor also has a hydrophobic residue (L147), and its mutation led to impaired Gq signaling (17).
We propose that M527 stabilizes the hydrophobic core of ICL2, thus positioning other aa in suitable positions for Gq-protein coupling. Support for this possibility can be derived from the inhibition of Gq activation by F525A, R528A, L529A, and D530A in the vicinity of M527. Furthermore, its selectivity for Gq, its failure to interact experimentally with ICL3, and its likely cytosolic orientation in modeling provide additional support for a direct interaction of the hydrophobic residue M527 with Gq upon activation. Subsequent studies will provide examinations of these hypotheses by homology modeling of TSHR/Gß
complexes and related experiments. This will allow additional understanding of the dual G protein coupling of TSHR and particularly of the selectivity of activation.
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Acknowledgments |
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Footnotes |
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First Published Online October 21, 2004
Abbreviations: ASU, Alanine substitution; ICL, intracellular loop; IP, inositol phosphate; TM, transmembrane domain; 7TMR, seven transmembrane-spanning receptor; TSHR, TSH receptor; wt, wild-type.
Received August 10, 2004.
Accepted for publication October 12, 2004.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits. J Biol Chem 278:35063–35070q coupling specificity. Mol Endocrinol 16:814–823This article has been cited by other articles:
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) or presence (
) of 100 mU/ml bovine TSH. cAMP levels were determined as described in Materials and Methods. Data are presented as the mean ± SEM of three independent experiments. B, Forty-eight hours after transfection, cells were labeled with 2 µCi/ml [myo-3H]inositol for 8 h and subsequently incubated in the absence (
