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Endocrinology Vol. 141, No. 10 3717-3722
Copyright © 2000 by The Endocrine Society

ARTICLES

Juxtamembrane Regions in the Third Intracellular Loop of the Thyrotropin-Releasing Hormone Receptor Type 1 Are Important for Coupling to Gq1

Friedrich Buck, Wei Wang, Sonke Harder, Claude Brathwaite, Thomas O. Bruhn and Marvin C. Gershengorn

Institut für Zellbiochemie und Klinische Neurobiologie (F.B., S.H., T.O.B.), Universitäts-Krankenhaus Eppendorf, D-20246 Hamburg, Germany; and Division of Molecular Medicine (W.W., C.B., M.C.G.), Department of Medicine, Weill Medical College and Graduate School of Medical Sciences of Cornell University, New York, New York 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Juxtamembrane residues in the putative third intracellular (I3) loops of a number of G protein-coupled receptors (GPCRs) have been shown to be important for coupling to G proteins. According to standard hydropathy analysis, the I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 amino acids from position-213 to position-263. We constructed deletion and site-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulated signaling activities. As expected, the effects of these mutations on TRH binding were small (less than 5-fold decreases in affinity). No effect on TRH-stimulated signaling activity was found in a mutant receptor in which the I3 loop was shortened to 16 amino acids by deleting residues from Asp-226 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 or from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In the region near transmembrane helix 6, single site-specific substitution of either Arg-261 or Lys-262 by neutral glutamine had little effect on signaling, but mutant TRH-Rs that were substituted by glutamine at both basic residues exhibited reduced TRH-stimulated activity. The reduced signaling activity of this doubly substituted mutant was reversed by over expressing the {alpha} subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRH RECEPTORS (TRH-Rs)2 are members of the G protein-coupled receptor (GPCR) family (1). A TRH-R complementary DNA (cDNA) was initially cloned from a mouse pituitary tumor (2) and subsequently from rat (3, 4, 5), human (6, 7), and chicken (8) cells; this receptor is now termed TRH-R type 1 (TRH-R1). A second type of TRH-R was cloned from rat brain (9, 10, 11). TRH-Rs signal via the phospholipase C-inositol 1,4,5-trisphosphate-1,2-diacylglycerol pathway mediated by the action of a pertussis toxin-insensitive G protein (12, 13) that for TRH-R1 was shown to be Gq/11 in rodent pituitary cells (14) and in Xenopus oocytes (15).

There is evidence for a key role of the I3 loops of GPCRs in coupling to G proteins (16). In particular, residues within the amino- and carboxyl-termini of the I3 loops affect this interaction. However, a consensus recognition motif for G protein coupling has not been identified. Indeed, the I3 loop is among the least conserved regions of GPCRs. TRH-Rs from all species investigated so far are very similar and contain the sequence Arg-Lys/Arg-Gln in their putative third intracellular (I3) loop near transmembrane helix six. These residues correspond to positions-261, 262, and 263 in the mouse TRH-R1 (mTRH-R1). It was previously shown that removal of residues from position-218 through position-263 in the I3 loop of mTRH-R1 formed a mutant receptor that did not signal in response to TRH (17). The focus of this study was to determine the residues of the I3 loop of TRH-R that are critical to receptor function by determining which residues are needed for TRH-stimulated signaling activities. We have deleted all but six residues in the amino-terminus and three residues in the carboxyl-terminus of the I3 loop of mTRH-R1 and made site-specific substitutions at positions-261, 262, and 263. All mutant receptors were tested for TRH-stimulated signaling in Xenopus oocytes, and for ligand binding and TRH-stimulated signaling in COS-1 and HEK 293 EM cells. We confirmed that the amino and carboxyl termini of the I3 loop are necessary for signaling and found that mTRH-R1s substituted by Gln at both Arg-261 and Lys-262 exhibited decreased TRH-stimulated signaling activities caused by decreased coupling to Gq.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
TRH was obtained from Bachem (Basel, Switzerland) or Sigma (St. Louis, MO). [3H][methyl-His]TRH ([3H]MeTRH) was from NEN Life Science Products (Boston, MA), Taq polymerase from Perkin-Elmer Corp. (Norwalk, CT), T7 RNA polymerase and restriction enzymes from Fermentas (Vilnius, Lithuania), deoxynucleotide triphosphates, and capping reagent from Amersham Pharmacia Biotech (Arlington Heights, IL). All other reagents were analytical grade.

Construction of mTRH-R1 mutants
Mutants of mTRH-R1 carrying deletions within the I3 loop were constructed by replacing the BstEII/BstEII fragment between bases 336 and 1047 of the receptor cDNA clone pBSmTRH-R (2) by a PCR fragment bearing the desired deletion. The PCR products were digested with BstEII and ligated into the BstEII cut pBSmTRH-R. For transfections into mammalian cells, deletions were introduced in the same way into the plasmid pCDM8mTRH-R, which contains the mTRH-R1 cDNA in the vector pCDM8 (2). The DNA sequences of all constructs were confirmed on both strands of the replaced fragment.

Receptor activity tests in oocytes
In vitro complementary RNA synthesis, Xenopus oocyte injections and electrophysiological measurements were performed as described (18). Measurements of the increase in membrane conductivity in voltage-clamped Xenopus oocytes injected with in vitro synthesized receptor cRNA and challenged with 10-8 M TRH were used as a rapid and sensitive assay for the ability of the mutant receptors to activate the inositol 1,4,5-trisphosphate-Ca2+ second messenger cascade. Because signal intensity has been shown to be a linear function of receptor density (19), the signal amplitude induced by a mutant receptor at a receptor density similar to that of WT receptor can be considered a valid measure of the relative ability to activate the signal transduction pathway in oocytes. To attempt to achieve similar receptor densities in mutant and WT receptor expressing oocytes, cRNA synthesis, injection and electrophysiological measurements were done for mutant and WT mTRH-Rs strictly in parallel. For each mutant mTRH-R1, at least three independent experiments were performed with each experiment using 10–15 oocytes.

Transfection of mammalian cells
COS-1 cells were maintained and transfected using the DEAE dextran method as described (2). HEK 293 EM cells, which express the Epstein-Barr nuclear antigen 1 and the human Class A macrophage scavenger receptor, were a generous gift of R. A. Horlick (Pharmacoepia, Cranbury, NJ). HEK 293 EM cells were maintained and transfected using calcium phosphate (20). The amount of plasmid DNA encoding receptors or the {alpha} subunit of Gq, a generous gift of F. C. Bancroft (Mount Sinai Medical Center, NY), was 2 µg/ml and the total DNA in all transfections of an experiment was made equal by adding "empty" plasmid.

Receptor binding assay
Binding experiments were performed with intact COS-1 and HEK 293 EM cells in monolayer at 37 C for 1 h. Equilibrium binding experiments were performed with increasing concentrations of [3H]MeTRH as described (2). Half-maximal inhibitory concentrations (IC50s) were measured in competition binding experiments using various doses of TRH and 1 nM [3H]MeTRH as radioligand as described (2). Curves were fitted by nonlinear regression analysis and Kd and IC50 values were calculated using the PRISM program (GraphPad Software, Inc., San Diego, CA).

Inositol phosphate formation
Stimulation of inositol phosphate (IP) second messenger formation by various doses of TRH was measured in COS-1 and HEK 293 EM cells prelabeled with myo-[3H]inositol as described (2). The stimulated activity of a mutant receptor in COS-1 and HEK 293 EM cells was defined as the maximum stimulation by TRH of IP second messenger formation in cells expressing mutant receptors compared with cells expressing WT mTRH-R1s in the same experiment. Comparisons were made only at levels of mutant receptor expression that were equal to or greater than those needed to produce maximal IP formation in cells expressing WT receptors. That is, there was no change in TRH-stimulated signaling activity in this range of receptor expression with changes in receptor number permitting assessment of the intrinsic TRH-stimulated activity.

Statistical analysis
Statistical analysis was performed by t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
According to standard hydropathy analysis, the I3 loop of mTRH-R1 is composed of residues from Phe-213 to Gln-263, thus comprising 51 amino acids (2) (Fig. 1Go). To determine the minimum number of residues in the I3 loop needed for signaling, a series of mutants with progressive deletions at the amino- and carboxyl-termini were studied in the Xenopus oocyte and COS-1 cell expression systems. There was little effect of deleting residues in the central portion of I3 on receptor expression, binding affinity or potency of TRH (Table 1Go). Mutant {triangleup}(226–260), in which 35 residues in the central portion of I3 from Asp-226 through Ser-260 were deleted creating a receptor with only 16 residues in I3, exhibited expression, binding and activity that were indistinguishable from WT. In general, the TRH-stimulated activities of the deletion mutants measured in transfected COS-1 cells correlated well with the results obtained in cRNA-injected oocytes. There were, however, a few exceptions. {triangleup}(226–262) and {triangleup}(226–263), in which 37 and 38 residues were deleted from the I3 loop including the residues at the carboxyl terminus, respectively, showed small losses in binding affinity and progressive decreases in TRH-stimulated activity in Xenopus oocytes but only {triangleup}(226–263) exhibited decreased TRH-stimulated activity in COS-1 cells. Shortening the I3 loop at the amino terminus in mutants {triangleup}(223–260) and {triangleup}(222–260) had little effect on affinity but led to progressive decreases in TRH-stimulated activity both in COS-1 cells and in Xenopus oocytes. Moreover, mutants {triangleup}(226–263) and {triangleup}(223–260), both of which contain thirteen residues in I3 but differ in the site of the deletion, displayed little change in affinity but markedly different TRH-stimulated activities: {triangleup}(226–263) was 25% as active as WT vs. {triangleup}(223–260) that was 75% as active as WT. The residues at the carboxyl terminus of the I3 loop (positions-260 through 263) are, therefore, required for optimal TRH-stimulated signaling by TRH-R.



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  		Figure 1. Schematic representation of mTRH-R1 showing the putative third intracellular loop from residues Phe-213 to Gln-263. The sequence deleted in {triangleup}(226–260) is indicated.

 

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  		Table 1. Binding and activation of WT and deletion mutant mTRH-R1s expressed in Xenopus oocytes and COS-1 cells

 
Because residues in the middle portion of the I3 loop were not important for receptor expression, binding, or signaling, we used the deletion mutant {triangleup}(226–260), which displayed similar activity and binding as WT, as a control to assess the role of residues at the juxtamembrane carboxyl terminal region of I3 (Table 2Go). Single site-specific mutations were made in residues Arg-261, Lys-262 and Gln-263 of {triangleup}(226–260). Mutants {triangleup}(226–260)/R261A and {triangleup}(226–260)/K262A exhibited binding and TRH activation profiles similar to {triangleup}(226–260). Evidence that position-261 and position-262 are important for receptor expression and function may be derived from mutants {triangleup}(226–260)/R261E and {triangleup}(226–260)/K262E that exhibit deficiencies when the positively charged residues were substituted by the negatively charged Glu. Both {triangleup}(226–260)/R261E and {triangleup}(226–260)/K262E exhibited high affinity binding but were expressed to lower levels than WT and displayed decreased TRH-stimulated signaling activities. In contrast, Gln-263 does not appear to be important because {triangleup}(226–260)/Q263E and {triangleup}(226–260)/Q263R exhibited levels of expression, binding affinities, and TRH-stimulated signaling activities similar to WT. Thus, substitution of Arg-261 or Lys-262 by neutral residues did not affect receptor function, whereas substitution by negatively charged residues decreased TRH-stimulated signaling activity.


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  		Table 2. Binding and activation of WT and I3 loop C-terminus mutant mTRH-R1s expressed in Xenopus oocytes and COS-1 cells

 
To determine whether any positive charge is needed at the carboxyl terminus of I3 for optimal TRH-stimulated signaling, both Arg-261 and Lys-262 were substituted by Gln in {triangleup}(226–260) ({triangleup}(226–260)/R261Q/K262Q) and in WT (R261Q/K262Q). The single substitution mutant TRH-Rs, {triangleup}(226–260)/R261Q and {triangleup}(226–260)/K262Q were expressed at the same levels as WT (data not shown) and exhibited similar levels of maximal signaling activity and TRH potencies (Table 3Go) as WT. Figure 2Go illustrates that R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q were expressed at levels between 35% and 65% of WT; {triangleup}(226–260) was expressed to a higher level in these experiments. The affinities of binding MeTRH were similar for all of these receptors. Kds were 0.98 (0.43–1.5) nM for WT, 0.68 (0.50–0.85) nM for {triangleup}(226–260), 1.0 (0.49–1.8) nM for {triangleup}(226–260)/R261Q/K262Q and 2.1 (0.45–3.7) nM for R261Q/K262Q. Thus, there were no differences in binding among these receptors. There were important differences with regard to TRH-stimulated signaling. In Xenopus oocytes, TRH-stimulated activity of {triangleup}(226–260)/R261Q/K262Q was reduced to 15% of WT (data not shown). In HEK 293 EM cells, both R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q exhibited reduced TRH-stimulated maximal signaling activities and decreased potencies (Table 3Go). The decreases in maximal stimulation appear to be caused by the lower levels of receptor expression. Similar findings were obtained in COS-1 cells (data not shown). In both COS-1 and HEK 293 EM cells, the potencies of TRH were reduced 6- to 10-fold in R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q compared with WT and {triangleup}(226–260). We used both cell lines to show that these differences were not a peculiarity of one mammalian cell line. The findings that there were no differences in the binding affinities of these receptors but differences in the potencies of TRH for signaling, were consistent with the idea that R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q were less efficiently coupled to G proteins than WT or {triangleup}(226–260). Indeed, using the equation for receptor efficiency derived by Ballesteros et al.3 (21), the efficiency of coupling for {triangleup}(226–260) was indistinguishable from WT, whereas that for {triangleup}(226–260)/R261Q/K262Q was 44% and that for R261Q/K262Q was 38% of WT.


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  		Table 3. Activation of R261Q or K262Q mutant TRH-Rs in HEK 293 EM cells

 


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  		Figure 2. Binding of [3H]MeTRH to intact HEK 293 EM cells expressing WT, {triangleup}(226–260), R261Q/K262Q or {triangleup}(226–260)R261Q/K262Q. Equilibrium binding of [3H]MeTRH to mTRH-R1s was performed as described in Materials and Methods. The data represent the mean ± SD of triplicate determinations in a representative of three experiments.

 
To test whether {triangleup}(226–260)/R261Q/K262Q and R261Q/K262Q exhibited reduced G protein coupling directly, we coexpressed the {alpha} subunit of Gq and these TRH-Rs. We used HEK 293 EM cells for these experiments because we could better control the ratio of Gq to TRH-Rs in these cells than in COS-1 cells. We predicted that increasing the number of G proteins would reverse the decreases in TRH potencies with R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q because the decreased G protein coupling efficiencies would be overcome. Figure 3Go illustrates the effects of overexpressing Gq {alpha} subunits on the potencies of and maximal responses to stimulation by TRH. The maximal responses of all four receptors were increased by overexpression of Gq {alpha} subunits; however, the increases were only 2-fold for WT and {triangleup}(226–260) but were 3-fold for R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q. More importantly, there were only small effects of overexpressing {alpha} subunits of Gq on the TRH potencies exhibited by WT and {triangleup}(226–260) receptors, whereas there were 5- to 10-fold increases in the TRH potencies of R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q. In the experiment illustrated in Fig. 3Go, the potencies without and with Gq {alpha} subunits were: 2.1 and 0.91 nM for WT; 1.2 and 0.41 nM for {triangleup}(226–260); 21 and 2.6 nM for {triangleup}(226–260)/R261Q/K262Q; and 26 and 5.2 nM for R261Q/K262Q. These findings are consistent with the idea that the two doubly mutated receptors exhibited reduced coupling to Gq compared with WT and {triangleup}(226–260) receptors.



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  		Figure 3. Effect of coexpressing {alpha} subunits of Gq with WT, {triangleup}(226–260), R261Q/K262Q or {triangleup}(226–260)R261Q/K262Q on TRH-stimulated signaling in HEK 293 EM cells. Signaling experiments were performed as described in Materials and Methods. The data are presented as % of the maximal response for WT or mutant receptors in the absence of exogenously coexpressed Gq {alpha} subunits. The data represent the mean ± SD of triplicate determinations in a representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, deletion and site-specific mutations within the I3 loop of the mTRH-R1 were constructed and used to probe the role of this domain in coupling to G proteins that activate the phospholipase C signaling pathway (1). We found that 35 residues from Asp-226 to Ser-260 in the midportion of the 51 amino acid sequence of the I3 loop could be deleted without changing receptor expression, binding, or TRH-stimulated signaling activity. Further deletion of residues at the amino or carboxyl termini of I3 caused significant losses in TRH-stimulated signaling. To determine which residues in the carboxyl terminal region of I3 were important in receptor function, we constructed site-specific mutants in this region. Our experiments with site-specific substitutions of Arg-261 and Lys-262 showed that the presence of at least one of these residues at either of these positions was necessary to maintain TRH-stimulated signaling activity. We suggest that a positively charged residue is needed in this region of the receptor to allow for efficient interaction with Gq. This interpretation derives from our findings (Fig. 3Go) that overexpression of {alpha} subunits of Gq reverses the loss in apparent potencies of TRH in cells expressing R261Q/K262Q and {triangleup}(226–260)/R261Q/K262Q. An additional role for these basic residues may be to interact with the negatively charged phospholipid head groups to anchor the receptor within the plasma membrane in a fully functional form.

These findings with TRH-R mutants are in agreement with observations made with other GPCRs for which it has been reported that residues in the juxtamembrane regions of the amino and carboxyl termini of I3 are important for G protein coupling (16, 22). In particular, hydrophobic residues at the amino and carboxyl termini of I3, which may form {alpha} helices, have been implicated in coupling to G proteins as have basic residues in the region near transmembrane helix 6 (23). In general, studies that have provided evidence in support of this idea, like the one reported herein, have involved receptor mutagenesis or the use of peptides derived from sequences found in this region of the receptor to inhibit G protein coupling. A more direct experiment was the use of site-directed spin labeling that showed that two residues in this region of bovine rhodopsin undergo increased mobility upon excitation by light (24). This increased mobility may allow more efficient coupling to transducin.

In general, the mutant mTRH-R1s that we studied exhibited similar TRH-stimulated activities in Xenopus oocytes and mammalian COS-1 cells. However, we observed divergent activities of a few mutant receptors in these two expression systems (Tables 1Go and 2Go). This was more obvious with the I3 loop carboxyl terminal site-specific mutant receptors than with the deletion mutants. For example, {triangleup}(226–260)/K262E and {triangleup}(226–260)/R261Q/K262Q exhibited only 15% of the TRH-stimulated activity of WT in Xenopus oocytes but were 54% to 60% as active in response to TRH in COS-1 cells. Whether these differences reflect important differences in coupling to G proteins in the two cell systems is not clear. Of note, the sequences of Gq/11 proteins are very similar in mammals and in Xenopus (25). Therefore, although Xenopus oocytes are an important model system for studies of GPCR function, in particular, where access to the intracellular milieu is critical, some caution must be exercised when relating findings in Xenopus oocytes to GPCR function in mammalian cells.

In conclusion, the juxtamembrane regions of the I3 loop, including one of the two carboxyl terminal basic amino acids, Arg-261 or Lys-262, of mTRH-R1 permits efficient coupling to Gq. On the other hand, phospholipase C-mediated signaling by TRH can be subserved by a receptor with an I3 loop of only 16 residues (in which 35 midportion residues were deleted). The roles of the amino acids in the midportion of the I3 loop in signaling via other pathways or in mTRH-R1 regulation are areas for future study.


   Footnotes
 
Address all correspondence and requests for reprints to: Marvin C. Gershengorn, Weill Medical College of Cornell University, 1300 York Avenue, Room A328, New York, New York 10021.

1 This work was supported by USPHS Grant DK-43036 (to M.C.G.). Back

2 The abbreviations used are: TRH, thyrotropin-releasing hormone; TRH-R, TRH receptor; mTRH-R1, mouse TRH-R type 1; GPCR, G protein-coupled receptor; I3, putative third intracellular loop; WT, wild-type mTRH-R1; {triangleup}(226–260), mTRH-R1 in which residues from Asp-226 through Ser-260 were deleted; R261Q/K262Q, mTRH-R1 in which Arg-261 and Lys-262 were substituted by Gln; {triangleup}(226–260)/R261Q/K262Q, {triangleup}(226–260) in which Arg-261 and Lys-262 were substituted by Gln (other mutant receptors are identified according to this scheme). Back

3 Receptor (coupling) efficiency Q = 0.5 x [(Kd + EC50)/EC50] x (Emax/Bmax) where Kd is the equilibrium dissociation constant, EC50 is the potency (concentration of agonist that gives a half-maximal effect), Emax is the maximal effect, and Bmax is the maximal binding capacity (receptor density). Back

Received April 20, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals