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Endocrinology Vol. 142, No. 3 1188-1194
Copyright © 2001 by The Endocrine Society

ARTICLES

Regulator of G Protein Signaling 4 Suppresses Basal and Thyrotropin Releasing-Hormone (TRH)-Stimulated Signaling by Two Mouse TRH Receptors, TRH-R1 and TRH-R21

Sönke Harder, Xinping Lu, Wei Wang, Friedrich Buck, Marvin C. Gershengorn and Thomas O. Bruhn

Institut für Zellbiochemie und klinische Neurobiologie (S.H., F.B., T.O.B.), Universität Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany; and Division of Molecular Medicine (X.L., W.W., M.C.G.), Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021-4896

Address all correspondence and requests for reprints to: Dr. Marvin C. Gershengorn, Weill Medical College of Cornell University, 1300 York Avenue, Room A328, New York, New York 10021-4896. E-mail: mcgersh{at}med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical with rat TRH-R2 and 50% identical with mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9, and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of G protein-coupled receptor activity under normal physiologic circumstances.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REGULATOR of G protein signaling (RGS) proteins are a family of more than twenty proteins that inhibit (or desensitize) receptors that transduce their signals by coupling to G proteins of the Gi, Gq, and G12/13 subfamilies (1, 2, 3). RGS proteins stimulate the GTP-hydrolyzing activities of G protein {alpha} (G{alpha}) subunits and thereby promote the conversion of G{alpha} from an active to an inactive state. [It appears that RGS proteins are effectors for G proteins also (4).] The specificity of RGS protein inhibition of G protein signaling in vivo is not understood and may differ from that observed in in vitro systems. Although several RGS proteins may inhibit Gq-mediated signaling activated by different receptors, it is not possible to predict whether a given RGS protein will inhibit signaling initiated by a specific receptor. RGS4 has been shown to interact with G{alpha}q leading to an increase in its GTPase activity and thereby dampen signaling by agonists that interact with some receptors that couple to Gq (5, 6). However, RGS4 apparently does not inhibit signaling by all Gq-coupled receptors as it has been reported, for example, that it does not affect signaling by the receptor for GnRH (7). Moreover, Xu and colleagues (8) showed that RGS4 inhibited agonist-stimulated, Gq-mediated signaling by different receptors expressed in the same cell with different sensitivities. To our knowledge, the effect of RGS4 on basal (agonist-independent) signaling by Gq-coupled receptors in intact cells has not been reported previously.

Two types of TRH-Rs have been cloned. TRH-R type 1 (TRH-R1) was originally cloned from a mouse thyrotropic pituitary tumor (9) and then from rat (10, 11, 12), human (13, 14, 15), chicken (16), and bovine (17) tissues. Up to the present, TRH-R type 2 (TRH-R2) has been cloned only from a rat brain stem-spinal cord complementary DNA (cDNA) library (18) and rat brain cDNA libraries (19, 20). We have shown that TRH-R1 and TRH-R2 bind agonists with indistinguishable affinities (20) and that TRH-R2, like TRH-R1, signals via the phosphoinositide pathway (20), most likely by coupling to a Gq subfamily member (21, 22). In contrast, the two receptor subtypes exhibit different tissue expression (18, 20) and TRH-R2 exhibits a much higher basal activity than does TRH-R1 (23). In this report, we describe the cloning of the mouse homolog of TRH-R2, which would be important for future studies of its physiology in intact animals, and test whether there is a differential sensitivity of TRH-R1 and TRH-R2 with regard to desensitization by RGS proteins. We found that TRH activation of both TRH-R types is similarly dampened by RGS4 but not by RGS7, RGS9, or GAIP. We show, moreover, that basal signaling by TRH-R2 is inhibited by RGS4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of TRH-R2 from mouse brain
Total RNA was isolated from mouse brain by acid guanidinium thiocyanate-phenol-chloroform extraction. Oligo (dT) primed cDNA was synthesized with SuperScript (Life Technologies, Inc.). Two rounds of PCR were necessary to amplify a 399-bp fragment that was distinct from mTRH-R1 but exhibited a high degree of homology with rTRH-R2. For the first round of PCR, degenerate oligonucleotide primers were: forward 5'-GTNGCNGCNGGNYTNCCNAAYA-3' and reverse 5'-CNARNGTNCKRTANGGCATCCA-3' with N corresponding to A, C, G or T, Y corresponding to C or T, K corresponding to T or G and R corresponding to A or G were employed. The primer design was based on homology within transmembrane domains 2 and 6 of known mammalian TRH-receptors corresponding to the amino acid sequences VAAGLPN and WMPYRTL, respectively, of mTRH-R1 (9). No distinct product with the expected size of 620 bp was obtained. Therefore, a second round of PCR using nested primers based on the amino acid sequences LRAQTVC and MLAVVVL, respectively, of rat TRH-R2 (18, 19, 20) was carried out. The sequences of these primers were: forward 5'-CTGAGAGCACAGACCGTGTG-3' and reverse 5'-CAACACAACCACGGCCAGCAT-3'. A product of approximately 400 bp was obtained, directly sequenced and cloned into a TOPO TA cloning vector (Invitrogen). This mTRH-R2 fragment was used as a probe to screen a mouse brain cDNA library (CLONTECH Laboratories, Inc.), however, positive clones were not obtained. Consequently, approximately 3 x 106 clones of a mouse genomic library (EMBL3; CLONTECH Laboratories, Inc.) were screened. Two positive clones were obtained and characterized. Phage DNA was prepared and directly sequenced according to a protocol published previously (24). Because both clones contained the 5' area of the mTRH-R2 gene stretching from the promoter region to the beginning of TM 6, 3' RACE (rapid amplification of cDNA ends; 5', 3' RACE kit, Roche Molecular Biochemicals, Mannheim, Germany) was employed to obtain the missing 3' coding region flanked by 3' UTR sequences (25). RACE products were cloned into the TOPO TA cloning vector (Invitrogen) and sequenced. Sequence assembly was carried out with SeqMan (DNAStar) and oligonucleotide primers located in the 5' and 3' untranslated regions were designed to amplify the full-length cDNA containing the entire coding regions with a mixture (4:1) of Taq and Pfu (Stratagene, La Jolla, CA) from mouse brain cDNA. Due to the low expression of TRH-R2 in mouse brain, two rounds of PCR were necessary to obtain a 1340-bp fragment containing the entire mTRH-R2 cDNA. The primers for the first round of PCR were 5'-CCTGGGTTCAATTCCCAGCACC-3' (forward) and 5'-ACCTCCCACCCAGGGTCCAGC-3' (reverse). The nested primers for the second round of PCR were 5'-CTTACCAAGGTCAAGGCCGG-3' (forward) and 5'-AGAGCGTTTGAGTGTCCTTCT-3' (reverse). The resulting DNA fragment of 1340 bp was cloned into the expression vector pcDNA3 (Invitrogen) downstream of the CMV promoter to generate plasmid pcDNA3-mTRH-R2.

Northern blot analysis of mTRH-R2 expression
The distribution of mTRH-R2 transcripts in tissues was studied by using a mouse multiple Northern blot (CLONTECH Laboratories, Inc.) containing 2 µg poly (A)+ messenger RNA (mRNA) isolated from heart, brain, spleen, lung, liver, skeletal muscle, kidney and testes. The blot was hybridized with a 32P labeled, randomly primed 400-bp cDNA fragment (see above: cloning of mTRH-R2) at 60 C overnight using express hybridization solution (CLONTECH Laboratories, Inc.). Following standard washes, the blot was exposed for 48 h and imaged using a Fujifilm BAS 1800 II phosphorimager (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Cell culture and transfections
HEK 293 EM cells (gift of Dr. Robert Horlick, Pharmacopeia, Cranbury, NJ) were grown in DMEM containing 10% FBS (Life Technologies, Inc.). On the day before transfection, the cells were seeded in 24-well dishes (30,000 cells/well). After 16 h, the medium was aspirated and the cells were transfected using calcium phosphate. The concentration of receptor-encoding plasmid DNA (mTRH-R1 or mTRH-R2) in transfection cocktails varied from 0.1 to 1 µg/ml. Where appropriate, 1 µg/ml pFR-Luc and 1 µg/ml pFA2-CREB (Stratagene) and/or 1 µg/ml plasmid encoding Myc-RGS4 (gift of Dr. Thomas M. Wilkie, University of Texas Southwestern Medical Center, Dallas, TX), FLAG-RGS7 (gift of Dr. Gerd Walz, Harvard Medical School, Boston, MA) or RGS9 or GAIP (gifts of Dr. Alfred Gilman, University of Texas Southwestern Medical Center, Dallas, TX) was added to the transfection cocktail. Total DNA was kept constant by adding empty plasmid. Mock transfections were performed without protein-encoding plasmid. The cells were exposed to the transfection cocktail for 6 h and were then incubated in DMEM containing 1% FBS for 16 to 24 h.

Measurement of TRH-R expression
Expression of mTRH-R1 and mTRH-R2 was measured as maximal binding of [3H]methyl-TRH in intact cell monolayers as described (26). The concentration of [3H]methyl-TRH was 0.1 to 10 nM. The data were analyzed using PRISM software (GraphPad Software, Inc., San Diego, CA).

Measurement of phosphoinositide hydrolysis
Acute stimulation of phosphoinositide hydrolysis by TRH was measured as accumulation of 3H-labeled inositol phosphates (IPs) over 60 min in the presence of 10 mM LiCl in myo-[3H]inositol labeled cells as described (26). Basal phosphoinositide hydrolysis was measured for the times indicated in the presence of 10 mM LiCl.

Assay of luciferase activity
Cells in 24-well plates were washed with PBS and lysed with 0.5 ml of lysis buffer (25 mM GlyGly, pH 7.8, 15 mM MgSO4.6H2O, 4 mM EGTA, 1 mM dithiothreitol, 1% Triton X-100). Cell lysates (0.025 ml) were combined automatically with 0.125 ml reaction buffer (25 mM GlyGly, pH 7.8, 15 mM MgSO4.6H2O, 4 mM EGTA, 1 mM dithiothreitol, 15 mM KH2PO4, 2 mM ATP) and 0.025 ml luciferin (0.4 mM; Sigma, St. Louis, MO) in reaction buffer and the luminescence was measured for 10 sec in a TR717 Microplate Luminometer (Tropix, Bedford, MA).

Assay of RGS4 expression
RGS4 expression was demonstrated by immunoblot using an anti-Myc antibody (6). Transfected cells were washed twice with PBS, and lysed with PBS containing 1% Triton X-100 and proteinase inhibitors. The lysate was placed in a bath sonicator for 15 min, transferred to a microfuge tube, and centrifuged at 12,000 x g for 30 min. The supernatant was diluted 1:1 with 2x Laemmli buffer, heated at 98 C for 10 min, and electrophoresed in a 12% gel. The proteins were transferred to a membrane, blocked with buffer containing 5% BSA, and incubated with 2 µg/ml 9E10 anti-Myc antibody (CLONTECH Laboratories, Inc.) for 1 h at room temp. The membrane was washed three times with PBS containing 0.1% Tween-20 and incubated with goat antimouse IgG conjugated to horseradish peroxidase (New England Biolabs, Inc.) for 1 h at room temp. The membrane was washed three times, the chemiluminescence reaction was for 1 min and the exposure was for 1 min.

Data analysis
Statistical significance was determined using Student’s t test with a probability criterion of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of mTRH-R2 from brain
Sequence analysis of the cDNA encoding a TRH-receptor different than TRH-R1 from mouse brain revealed an open reading frame for a 42.0-kDa protein containing 382 amino acids. The protein exhibited 92% identity with the recently cloned rTRH-R2 (18, 19, 20). Within transmembrane domains, mTRH-R2 and rTRH-R2 were 92.8% identical (Fig. 1Go). Four residues in putative transmembrane helices (TMs) 3, 6, and 7, which have been shown to directly bind TRH in mTRH-R1 (27), were fully conserved in mTRH-R2 (Tyr103 and Asn107 in TM 3, Tyr270 in TM 6 and Arg294 in TM 7). The relative homology between TRH-receptors of subtype 1 vs. subtype 2 was similar in mouse and rat. mTRH-R1 compared with mTRH-R2 and likewise, rTRH-R1 compared with rTRH-R2, exhibited an identity of approximately 50%. Within a subtype, TRH-receptors of mouse and rat were >90% identical.



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  		Figure 1. Alignment of the amino acid sequences of TRH-R2 from mouse (m) and rat (r). *, Amino acid identity between mTRH-R2 and rTRH-R2. Arrows indicate the position of introns within the coding region.

 
Organization of the mTRH-R2 gene
Sequencing of 5890 bp of an EMBL 3 clone using a gene walking approach revealed the presence of two introns located within the coding region of the mTRH-R2 gene. One intron was located at the end of TM 3 and another positioned at the beginning of TM 6 (or at the end of the third intracellular loop) (Fig. 2Go, see also Fig. 1Go). Because the mTRH-R2 lambda clone ended within this second intron upstream of the sequence encoding TM 6, we were not able to determine the splice acceptor sequence and the size of the second intron. cDNA sequences 3' of TM 6 could not be verified at the genomic level due to the 5' orientation of the clone. However, cDNA-sequences 5' of TM 6 were verified at the genomic level. Figure 2Go also depicts sequences of the exon/intron boundaries.



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  		Figure 2. Organization of the mTRH-R2 gene. Genomic and mRNA sequences are represented schematically. Transmembrane domains are indicated as black bars. The lambda clone contained the 5' region of the mTRH-R2 gene including the reading frame up to the end of the third intracellular loop (I 3), interrupted by an intron located at the end of TM 3. This lambda clone ended within the second intron at the end of the third intracellular loop and, therefore, did not reveal the structure of the mTRH-R2 gene 3' of the second intron. The coding region 3' of TM 6 that was cloned from cDNA must be present on a putative exon 3 indicated by stippled lines.

 
Northern Blot analysis of mTRH-R2 expression
Northern blot analysis of poly (A)+ selected RNA isolated from several tissues including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testes, but not including pituitary, revealed the presence of mTRH-R2 mRNA only in brain tissue. The apparent size of this mRNA was approximately 9.5 kb (Fig. 3Go).



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  		Figure 3. Northern blot analysis of mTRH-R2 expression. A mouse multiple Northern blot (CLONTECH Laboratories, Inc.) containing 2 µg poly (A)+ mRNA isolated from the tissues indicated above the x-ray image was hybridized with a 32P labeled, randomly primed 400 bp mTRH-R2 probe. A prominent band of approximately 9.5 kb was exclusively observed in brain.

 
Functional expression of mTRH-R2 in HEK 293 EM cells
The affinity of mTRH-R2 for MeTRH was indistinguishable, Kd = 1.8 (0.9–2.7) nM, from that of mTRH-R1, Kd = 1.8 (0.7–2.8) (data not shown). Similarly, in competition binding experiments, the calculated affinities of the two receptors for TRH were not different: Ki = 4.4 (2.5–8.0) nM for mTRH-R2 and 8.5 (5.0–15) nM for mTRH-R1 (Fig. 4AGo). When a series of TRH analogs was tested in binding experiments, no differences in the binding affinities of mTRH-R2 and mTRH-R1 were observed (data not shown). Finally, TRH stimulated inositol phosphate (IP) formation similarly in cells expressing mTRH-R2, EC50 = 0.34 (0.19–0.60), compared with cells expressing mTRH-R1, EC50 = 0.59 (0.32–1.1) (Fig. 4BGo).



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  		Figure 4. Effects of RGS4 on mTRH-R1 and mTRH-R2 binding and expression and TRH-stimulated signaling in HEK 293 EM cells. A, Competition binding of [3H]MeTRH by unlabeled TRH. Binding of [3H]MeTRH to mTRH-R1 or mTRH-R2 in the absence of unlabeled TRH was set at 100%. The binding affinity for TRH of the two mTRH-Rs was tested with or without coexpression of RGS4. The data represent the mean of triplicate determinations in two experiments. B, TRH stimulated inositol phosphate formation. Dose-response curves were generated in cells expressing either mTRH-R1 or mTRH-R2 without or with coexpression of RGS4. Data were normalized so that maximal responses were set at 100%. The data represent the mean of triplicate determinations in two experiments. Nonlinear regression analyses and curve fitting were performed with PRISM software (GraphPad Software, Inc.).

 
Effects of RGS proteins on basal and TRH-stimulated signaling
Cells transfected with plasmid encoding RGS4 and either mTRH-R1 or mTRH-R2 expressed readily measurable levels of RGS4 (see below). Coexpression of RGS4 with mTRH-Rs did not affect their affinities for TRH (Fig. 4AGo) nor, more importantly, consistently affect the level of expression of mTRH-R2 or mTRH-R1. In the experiments illustrated in Fig. 4Go, the maximal binding was 2230 (1890–2580) dpm/well and 2390 (2130–2650) for mTRH-R1 without and with RGS4, respectively, and was 1530 (1410–1650) and 1530 (1350–1720) for mTRH-R2 without and with RGS4, respectively. However, coexpression of RGS4 decreased TRH-stimulated IP production transduced by mTRH-R1 or mTRH-R2 by 65% to 75% (Fig. 4BGo).

To determine whether the effect of RGS4 was specific, we measured the effects of three other RGS proteins, GAIP, RGS7, and RGS9. Figure 5Go illustrates the results of experiments in which maximal stimulation of IP production by TRH (1 µM) in cells expressing one of the RGS proteins and mTRH-R1 or mTRH-R2 was assessed. The data are plotted as IP production vs. level of receptor expression. A linear relationship is evident when the data from cells transfected with mTRH-R1 alone, mTRH-R1 and GAIP, mTRH-R1 and RGS7, and mTRH-R1 and RGS9 are included. These data show that coexpression of these RGS proteins does not inhibit mTRH-R1 signaling. In contrast, the levels of TRH-stimulated IP production are significantly lower in cells expressing mTRH-R1 and RGS4. A similar set of data are shown for mTRH-R2 also. These data show that the effect of RGS4 is specific and that other RGS proteins do not affect mTRH-R1 or mTRH-R2 signaling.



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  		Figure 5. Effects of RGS proteins on TRH-stimulated inositol phosphate formation in cells expressing mTRH-R1 or mTRH-R2. Cells expressing mTRH-R1 (R1) alone, mTRH-R1 and RGS7 (R1/RGS7), mTRH-R1 and RGS9 (R1/RGS9), mTRH-R1 and GAIP (R1/GAIP), mTRH-R2 (R2) alone, mTRH-R2 and RGS7 (R2/RGS7), mTRH-R2 and RGS9 (R2/RGS9), or mTRH-R2 and GAIP (R2/GAIP) were stimulated by 1 µM TRH. The data represent the mean ± SD of triplicate samples in two experiments; the SD fell within the symbol in the cases where no error bars are presented. All values for R1 fell within the 95% confidence limits of the R1 regression line except the values for R1/RGS4 (filled squares). All values for R2 fell within the 95% confidence limits of the R2 regression line except the values for R2/RGS4 (unfilled squares).

 
Basal signaling was assessed by two different methods: 1) measurement of IP production, which is a proximal response after TRH-R activation; and 2) stimulation of gene transcription using the reporter gene luciferase, which is distal response but is more sensitive (23, 26). Figure 6AGo shows that easily detectable levels of RGS4 are present in cells transfected with the Myc-RGS4 plasmid. The levels of RGS4 varied from one experiment to another but RGS4 expression was measurable in all experiments in which Myc-RGS4 was transfected. Figure 6BGo illustrates a time course experiment in which basal IP production was measured in the same cells expressing mTRH-R1s or mTRH-R2s as used in Fig. 6AGo. As shown previously (26), there was no measurable basal IP production in cells expressing mTRH-R1s. In contrast, there was a readily measurable, constant rate of basal IP production in cells expressing mTRH-R2s. Moreover, basal IP production caused by mTRH-R2 was inhibited by 88 ± 8.2% in cells expressing RGS4 whereas no such effect could be detected in cells expressing TRH-R1.



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  		Figure 6. Effects of RGS4 on basal inositol phosphate formation in cells expressing mTRH-R1 or mTRH-R2. A, Immunoblot demonstrating expression of RGS4 in cells cotransfected with mTRH-R1 or mTRH-R2. B, Effect of RGS4 on basal signaling by mTRH-R1 or mTRH-R2. The data represent the mean ± SD of triplicate samples in the same representative experiment illustrated in Fig. 6AGo.

 
A more definitive way of demonstrating basal signaling by a receptor is to show that there is a direct correlation between signaling activity and the level of receptor expression. Figure 7Go illustrates this correlation for mTRH-R1 and mTRH-R2. As shown above, cells expressing mTRH-R1s did not exhibit any measurable basal IP production, whereas mTRH-R2 stimulated basal IP production in a receptor level-dependent manner (Fig. 7AGo). Basal IP production stimulated by mTRH-R2 was inhibited by coexpression of RGS4 by 50% to 60%. Using the more sensitive reporter gene assay, both mTRH-R1 and mTRH-R2 were shown to signal basally; the basal signaling activity of mTRH-R2 was 3.6- to 5.9-fold higher than that of mTRH-R1 (Fig. 7BGo). Using the reporter gene assay, coexpression of RGS4 inhibited basal signaling of mTRH-R2 by 37–57%. Although it was difficult to quantify due to the low level of basal signaling by mTRH-R1, coexpression of RGS4 appeared to inhibit the basal activity of mTRH-R1 also (Fig. 7BGo).



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  		Figure 7. Effect of RGS4 on basal signaling by mTRH-R1 and mTRH-R2. A, Receptor expression levels determine the effect of RGS4 on basal signaling by TRH-R as assessed by inositol phosphate formation. B, Correlation of receptor expression levels and basal signaling by mTRH-R1 and mTRH-R2 as assessed by a reporter gene assay. Basal signaling of mTRH-R1 and mTRH-R2 was determined as receptor density-dependent stimulation of inositol phosphate production (A) or reporter gene transcription (B). Reporter gene activity was recorded as luciferase activity in relative light units (RLU). In (B), the effect of RGS4 on basal signaling of TRH-receptors was determined by coexpressing reporter genes together with TRH-receptors and RGS4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We cloned a cDNA encoding mTRH-R2 from mouse brain. mTRH-R2 and rat TRH-R2 exhibit a high degree of homology at the amino acid level. Similar to the rat homologs, mTRH-R2 and mTRH-R1 bind TRH with indistinguishable affinity. This finding is not surprising because the four amino acid residues in putative TMs 3, 6, and 7, which have been shown to directly bind TRH in mTRH-R1 (27), were fully conserved in mTRH-R2. mTRH-R2 signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. Similar findings were reported for the rat homologs (23).

Three groups have previously cloned cDNAs for rat TRH-R2 (18, 19, 20), but the structure of its gene has not been reported. We found that the mTRH-R2 gene contains at least two introns within the coding sequence. One intron interrupts the sequence that encodes the end of putative TM 3 and the second intron interrupts the sequence that encodes the carboxyl end of the third intracellular loop. This is different from the structure of TRH-R1. Specifically, there is no intron in the TRH-R1 gene within the sequence that encodes the region at the end of TM 3 in any species that has been reported. The TRH-R1 gene, like the gene for mTRH-R2, was shown to contain an intron within the coding sequence at the carboxyl end of the third intracellular loop in humans (28) and in cows (17). Although it was reported that there is no intron at the end of the sequence that encodes the third intracellular loop in rats (11) and mice (29), we have found an intron in this location in the mTRH-R1 and rTRH-R1 genes (unpublished observations). The fact that the mTRH-R2 gene contains both a distinct intron located at the end of TM 3 and the conserved intron located at the end of the third intracellular loop suggests that TRH-receptor genes of the subtype 1 and 2 are derived from a common ancestor. This is consistent with the view that genome doublings occurred during vertebrate evolution (30, 31). Sequence divergence following gene duplication may be responsible for the genesis of TRH-R-subtypes as well as those of other G protein-coupled receptors (30).

The gene of TRH-R1 in rodents contains another intron within the sequence that encodes the carboxyl tail. In rats, there is a retained intron within this sequence that yields two rat TRH-R1 isoforms with different carboxyl tails via alternative splicing (11). In mice, the TRH-R1 gene contains an intron within the distal end of the carboxyl tail that allows two TRH-R1 isoforms to form via alternative splicing also (29). Our clones of the mouse TRH-R2 gene did not include sequences 3' of the intron after the exon encoding the third intracellular loop and, therefore, we have not determined whether there is an intron within the sequence that encodes the carboxyl tail in the mTRH-R2 gene.

Studies of the effects of RGS proteins on TRH-Rs have not been previously reported. We measured the effects of several RGS proteins on signaling by mTRH-R1 and mTRH-R2. RGS4, but not RGS7, RGS9 or GAIP, inhibited signaling by both mouse TRH-Rs. It is of note that RGS4 is expressed at high levels within the brain, especially in some areas like the paraventricular and mamillary nuclei of the hypothalamus and the olfactory cortex (32) where both TRH-R1 and TRH-R2 are expressed (20). RGS4 is also one of the most thoroughly studied RGS proteins and is an effective inhibitor of G{alpha}q activation of phospholipase C (5, 6); TRH-Rs signal via the phosphoinositide pathway by coupling to a Gq subfamily member (21, 22). Moreover, RGS4 inhibition of Gq-activated IP production was found to be receptor selective (8), apparently because its N-terminal domain can interact differently with different receptors (33). We, therefore, sought to determine whether RGS4 would affect mTRH-R1 and mTRH-R2 signaling and whether it would affect these receptors differently. We found that RGS4 effectively dampened TRH-stimulated IP production mediated by both mTRH-R1 and mTRH-R2 without having any effect on receptor binding or expression. More interestingly, we found that the marked basal signaling activity of mTRH-R2 was inhibited by RGS4. Although inhibition of Gq-mediated basal signaling might have been predicted, the effect of RGS4 on basal IP production has not, to our knowledge, been reported previously. The effect of RGS4 to inhibit basal signaling by mTRH-R2 may represent an important physiologic mechanism of regulation.

In conclusion, we have cloned the mTRH-R2 and delineated a gene structure that is different from that of TRH-R1. From a functional perspective, our finding that the marked basal signaling of mTRH-R2 can be inhibited by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of mTRH-R2 that might occur with other basally signaling G protein-coupled receptors under normal physiologic circumstances.


   Acknowledgments
 
We thank Dr. Thomas M. Wilkie for the plasmid encoding Myc-tagged RGS4, Dr. Gerd Walz for the plasmid encoding FLAG-RGS7, and Dr. Alfred Gilman for the plasmids encoding RGS9 and GAIP.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grant BR 794/2–3 and 2–4 to TOB and by USPHS Grant DK-43036 (to M.C.G.). Data deposition: DNA sequences reported in this paper have been deposited into the GenBank database under Accession Number AF283762 for the cDNA sequence of mTRH-R2 and under Accession Number AF283763 for the genomic DNA sequence of mTRH-R2. Back

Received October 10, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dohlman HG, Thorner J 1997 RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem 272:3871–3874[Free Full Text]
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