help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, Y.
Right arrow Articles by Gershengorn, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, Y.
Right arrow Articles by Gershengorn, M. C.
Endocrinology Vol. 143, No. 8 2886-2892
Copyright © 2002 by The Endocrine Society

ARTICLE

Correlation between Basal Signaling and Internalization of Thyrotropin-Releasing Hormone Receptors: Evidence for Involvement of Similar Receptor Conformations

Yuhua Sun and Marvin C. Gershengorn

The Division of Molecular Medicine, Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021; and the Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-8029

Address all correspondence and requests for reprints to: Marvin C. Gershengorn, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 50 South Drive, MSC 8029, Bethesda, Maryland 20892-8029. E-mail: . marving{at}intra.niddk.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that rat thyrotropin-releasing hormone (TRH) receptor type 2 exhibits higher basal signaling activity and internalizes more rapidly upon agonist binding than rat TRH receptor type 1. The mouse TRH receptor type 2 (mR2) was recently cloned and, similar to its rat homolog, shows a higher basal signaling activity than mR1. Taking advantage of the high degree of sequence homology between mR1 and mR2, we used chimeras/mutants of these receptors to gain insight into the properties of the receptors that influence internalization and basal signaling. Chimeric receptors that have the mR1 extracellular and transmembrane domains with the carboxyl terminus and intracellular loops of mR2 (R1/R2-tail; R1/R2-I3,tail; R1/R2-I2,3,tail; R1/R2-I1,2,3,tail) exhibited internalization rates and basal activities that were similar to that of mR1. In contrast, a chimeric receptor with the extracellular and transmembrane domains of mR2 and the carboxyl terminus of mR1 exhibited the more rapid internalization rate and higher basal signaling activity characteristic of mR2. We showed previously that mutation of a highly conserved tryptophan to alanine caused mR1 to exhibit a high basal signaling activity and rapid internalization rate. In contrast, mutation of this tryptophan to alanine in mR2 decreased the rate of internalization and inhibited basal signaling activity. The rates of receptor internalization did not correlate with the binding affinities, coupling efficiencies, or potencies of the receptors. Thus, we observed that receptors with more rapid internalization rates showed relatively higher basal signaling activities, whereas receptors with lower basal signaling activities showed slower internalization rates. These data suggest that similar receptor conformations are required for productive coupling to signaling G proteins and to proteins involved in internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECEPTOR INTERNALIZATION IS a common response that follows agonist binding to G protein-coupled receptors (GPCRs) and appears to modulate several aspects of receptor function such as termination of signaling, activation of MAPKs, resensitization, and down-regulation (1, 2, 3, 4). Although components of the internalization pathway of several GPCRs have been documented, the structural motifs within GPCRs that mediate internalization are not understood in most receptors. A number of studies have shown that the intracellular carboxyl terminus in many GPCRs is important for internalization because truncations or mutations of the amino acid residues in this region impaired receptor endocytosis (5, 6, 7, 8, 9, 10, 11, 12). Other studies indicated that mutations in the intracellular loops of the human GnRH receptor (GnRH R) (13) and the human and rat lutropin/choriogonadotropin receptor (LH R) (14), the extracellular domain of the human LH R (15, 16), and the transmembrane region of the FSH receptor (FSH R) (17) are capable of affecting receptor internalization.

Studies on mouse LH (18), ß2-adrenergic (19), and m3 muscarinic receptors (20) have shown that weak partial agonists induced slower rates of receptor internalization than full agonists. In addition, mutations of LH (21), ß2-adrenergic (22), and thyrotropin-releasing hormone (TRH) receptors (TRH R) (23) that impaired signaling activities also impaired agonist-induced receptor internalization. Therefore, it has been proposed that receptor activation is required for agonist-induced internalization. However, different conclusions have been drawn about receptor activation and internalization based on evidence that some mutations of the ß2-adrenergic receptor (24) and type 1 angiotensin II receptor (25, 26) caused impairment of signaling activity but did not affect internalization. Yeast {alpha}-factor (27), mammalian ß2-adrenergic (28), and mouse TRH Rs (29) are capable of internalization in the absence of G proteins. In addition, antagonist-bound type 1 angiotensin receptors (26) and antibody-bound TRH R (30) have also been shown to undergo internalization. More recent experiments conducted on signaling-impaired and phosphorylation-deficient mutants of the human LH R suggested that phosphorylation and activation play a redundant role in agonist-induced internalization (31), and the association of nonvisual arrestins, which is necessary for internalization, with human LH R depends more on receptor activation than receptor phosphorylation (32). In addition, studies of rat FSH Rs have demonstrated that agonist-induced activation and phosphorylation are not required for internalization, whereas the interaction of the rat FSH R with a nonvisual arrestin is essential for internalization (33).

TRH receptors (TRH Rs) are members of the rhodopsin/ß-adrenergic receptor subfamily of GPCRs (34). The binding of TRH activates signaling primarily via G-proteins of the G{alpha}q/11 family (35, 36, 37). Like other GPCRs, binding of agonists to TRH Rs triggers internalization of the agonist-receptor complex, most likely via the ß-arrestin mediated clathrin pathway (29, 30, 38, 39). The internalized TRH Rs are rapidly recycled to the plasma membrane, with only a fraction of the internalized TRH Rs targeted to a degradation pathway (38). Several mutations of TRH R1 have been shown to be defective in internalization, including receptors with mutations in the second transmembrane helix (TM 2), the third intracellular loop (I3), and a truncated receptor missing the carboxyl terminus (6, 23, 30, 38). Coupling to G protein is not sufficient to cause internalization (23). There are contradictory conclusions about whether receptor-G protein coupling is necessary for TRH R internalization (23, 29, 30, 40).

Two types of TRH Rs have been cloned. The cDNA of TRH R1 was initially cloned from mouse (41) and subsequently from rat (42, 43, 44), human (45, 46), chicken (47), and bovine tissues (48). Type 2 TRH R (TRH R2) was recently cloned from rat (49, 50, 51) and mouse (52). Amino acid sequence alignments of the two types of TRH Rs from the same species reveal a 50% overall identity. Despite their similarities in binding affinities for agonists and intracellular signaling pathways (51), TRH R1 and R2 exhibit several marked differences. First, unlike TRH R1, which is expressed in lactotrophs and thyrotrophs of the anterior pituitary, TRH R2 mRNA is extensively distributed in the brain, particularly in the cerebral cortex, with less abundance in the anterior pituitary (50, 51). Second, TRH R2 has a much higher basal signaling activity than TRH R1 (51, 52, 53). Third, rat TRH R2 (rR2) is more rapidly internalized and more markedly down-regulated after binding TRH than rR1 (51). Because TRH R2 is widely distributed in the brain, the high basal signaling activity and rapid agonist-induced internalization of TRH R2 may play an important role in mediating the function of TRH in the brain.

The focus of this study was to identify domains/residues of mouse receptors that influence their rates of internalization. We constructed mutant receptors in which we shifted the carboxyl terminus of mR1 and mR2, constructed chimeras containing the extracellular and transmembrane (E/TM) domains of mR1 and various intracellular domains of mR2, and made point mutations of mR1 and mR2. All receptors were tested for ligand binding, basal and stimulated signaling, and agonist-induced internalization in transfected HEK (human embryonic kidney) 293 EM (Epstein-Barr macrophage scavenger receptor) cells. The results showed that changes in the intracellular domains of mouse receptors that did not change their basal signaling activity had small effects on agonist-induced receptor internalization, whereas mutation of a highly conserved tryptophan residue in transmembrane helix 6 (TM6) that affected basal signaling greatly affected the rate of agonist-induced receptor internalization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[3H][methyl-His]TRH ([3H]MeTRH) was purchased from NEN Life Science Products (Boston, MA). Myo-[3H]inositol was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Restriction endonucleases, Vent DNA polymerase and deoxynucleotides were obtained from New England Biolabs, Inc. (Beverly, MA). DMEM and fetal bovine serum were purchased from Sigma (St. Louis, MO). The mammalian expression vector pcDNA3.1 was from Invitrogen (Carlsbad, CA). All reagents were analytical grade.

DNA
The full-length, mR1 in pMT4 (54) was subcloned into the pcDNA3.1 vector. The full-length mR2 in pcDNA3 was a gift from Dr. Thomas Bruhn (52). These plasmids were used as templates for mutagenesis. Plasmid pCDM8 containing an insert encoding mR1 with Trp at position 279 mutated to Ala (R1-W279A) was constructed as previously described (55). Overlapping PCR was used to generate fragments containing R2-W267A, R1/R2-tail, R2/R1-tail. The fragments were digested with EcoR I and NotI and then subcloned into pcDNA3.1. To construct R1/R2-I3 and R1/R2-I3,tail, three DNA fragments, which were obtained as follows, were ligated together. The mR1 or R1/R2-tail in pcDNA3.1 was digested with BstEII, and the resulting 5.9-kb and 711-bp fragments were purified by QIAGEN gel extraction kit (QIAGEN, Valencia, CA). The 711-bp fragment was then digested with MscI, and a 439-bp fragment was extracted and purified. The I3 of mR2 was generated by PCR using mR2 in pcDNA3 as template, and digested with BstEII and McsI. The 5.9-kb, 439-bp, and I3 of mR2 were subsequently ligated to yield plasmids encoding the chimeric receptors (R1/R2-I3 and R1/R2-I3,tail). To generate R1/R2-I2,3,tail and R1/R2-I1,2,3,tail, overlapping PCR was employed using R1/R2-I3,tail as template. The R1/R2-I1,2,3,tail receptor was obtained the same way using R1/R2-I2,3,tail as template.

Cell culture and transfections
HEK 293 EM cells were grown in DMEM containing 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). 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 in transfection cocktails varied from 0.1–2 µg/ml. Where appropriate, 1 µg/ml pFR-Luc and 1 µg/ml pFA2-CREB (cAMP-response element binding protein) (PathDetect In Vivo Signal Transduction Pathway trans-Reporting System, Stratagene, La Jolla, CA) was added to the transfection cocktail. The pFA2-CREB encodes a chimeric protein of the yeast Gal4 binding domain and the activation domain of CREB, and the pFR-Luc contains yeast Gal4 binding elements upstream of the luciferase gene. Total DNA was kept constant by adding empty plasmid. Mock transfections were performed with empty plasmid. The cells were exposed to the transfection cocktail for 6 h and were then incubated in DMEM containing 1% fetal bovine serum for 16–24 h.

Measurement of TRH R expression
Expression of mR1 and mR2 was measured as maximal binding of [3H]MeTRH in intact cell monolayers as described (56). The concentration of [3H]MeTRH was 0.1–10 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 (56). 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) in reaction buffer and the luminescence was measured for 10 sec in a TR717 Microplate Luminometer (Tropix, Bedford, MA).

Assay of agonist-induced receptor internalization
Internalization of TRH Rs was measured as specifically bound [3H]MeTRH that was resistant to acid wash (6). At the end of the incubation with 2 nM [3H]MeTRH for the time shown, free ligand was removed by aspirating the binding buffer and washing the cells with 2 ml ice-cold buffer. Cells were then exposed to 1 ml 50 mM glycine (pH 3.5), 0.5 M NaCl for 3 min at 4 C, and then washed with 2 ml binding buffer twice. Acid-resistant [3H]MeTRH was counted and compared with total specific binding. Internalized receptors were presented as the fraction of acid-resistant [3H]MeTRH-bound receptors divided by total [3H]MeTRH-bound receptors.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Roles of the carboxyl termini and intracellular loops in agonist-induced receptor internalization
The rates of agonist-induced internalization of many GPCRs are greatly affected by alterations in their carboxyl termini. In a previous report (6), we characterized two domains within the C-tail of mR1 that affect agonist-induced internalization. Truncations of the C-tail, or mutations of two cysteine residues in this region impaired agonist-induced internalization of mR1. The intracellular loops of many GPCRs have also been shown to play a role in agonist-induced internalization (13, 14, 23) and may interact with proteins involved in internalization (2). We began to examine whether changes in the carboxyl termini or intracellular loops of mR1 and mR2 could alter the rate of internalization. We exchanged the carboxyl terminus between mR1 and mR2. We also shifted the third, second, and first intracellular loop of mR2 sequentially (I3, I2, I1, respectively) to the chimeric receptor R1/R2-tail. The structures of these chimeric receptors are illustrated in Fig. 1Go. As shown in Fig. 2Go, the chimeric receptors exhibited agonist-induced internalization in a fashion similar to the receptor from which the E/TM domains were derived: mR2 (t1/2 = 0.1 ± 0.07 min) and R2/R1-tail (t1/2 = 0.3 ± 0.08 min) internalized more rapidly than the wild-type mR1 (t1/2 = 1.0 ± 0.2 min) and the chimeric receptors with E/TM domains of R1 and various portions of R2’s intracellular domains (t1/2 = 1.0 ± 0.2 min for R1/R2-tail; t1/2 = 1.0 ± 0.1 min for R1/R2-I3,tail; t1/2 = 1.0 ± 0.2 min for R1/R2-I1,3,tail). The internalization rates of R1/R2-I2,3,tail and R1/R2-I1,2,3,tail were similar or slower than mR1 (not shown).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Two-dimensional topologies of mR1 and mR2 chimeric receptors. The domains from mR1 and mR2 are shown in solid and dash, respectively.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Receptor internalization in HEK 293 EM cells transiently expressing wild-type (R1 and R2) and chimeric receptors. Transfected cells were incubated with 2 nM [3H]MeTRH at 37 C for the times indicated and specific and acid-resistant binding were measured as described in Materials and Methods. Results are expressed as mean ± SD of assays performed in triplicate in a representative experiment.

 
Internalization rates are correlated with basal activities of the receptors
We compared the basal activities of the more rapidly internalized receptors (mR2 and R2/R1-tail) with those of receptors that showed similar internalization rates to mR1 (R1/R2-tail, R1/R2-I3 and R1/R2-I3,tail). Basal signaling was estimated as the effects of increases in the number of expressed receptors (produced by varying the amount of plasmid in the transfection cocktail) on basal IP accumulation (Fig. 3AGo). The slopes of the lines describing the relationships between receptor expression and basal activities fell into two groups: for receptors displaying a rapid internalization (mR2 and R2/R1-tail), the slopes were significantly greater than zero, indicating that these receptors were constitutively active. For receptors displaying similar internalization rates as mR1, the slopes were near zero, indicating that these receptors were not measurably basally active in this assay. These observations were confirmed by using a luciferase assay in which basal activity is assessed as induction of transcription of the luciferase reporter gene (Fig. 3BGo), a system more sensitive than IP formation (53, 56). The receptors fell into the same two groups using this assay as with measurement of IPs. Thus, of the receptors that were tested in the present study, the rates of agonist-induced internalization were closely correlated with their basal activities; receptors with higher agonist-induced internalization rates showed greater basal activities. The internalization rate of a receptor was not correlated with its affinity, potency, or coupling efficiency assessed as the ratio of potency to affinity. As shown in Table 1Go, receptors that displayed different dissociation constants (Kds), half-maximally effective concentrations (EC50s), or maximal binding capacities, but the same basal activities showed similar internalization rates. Lastly, rapidly internalized receptors with higher basal activities responded to TRH stimulation to a lesser extent than the more slowly internalized receptors with lower basal signaling activities that showed greater responses to TRH stimulation (Table 1Go).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Basal IP second messenger formation and reporter luciferase activity as a function of wild-type or chimeric receptor expression levels. HEK 293 EM cells were cotransfected with plasmid containing pFA2-CREB, pFR-Luc, and various amounts of plasmid encoding wild-type or chimeric/mutant mRs. Cells were assayed for IP formation (A) and luciferase activity (B) as described in Materials and Methods. Results are expressed as mean ± SD of assays performed in triplicate in a representative experiment.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Internalization rates, affinities, maximal binding capacities, potencies, and fold increases in TRH-stimulated signaling activities of wild-type (R1 and R2) and chimeric/mutant receptors

 
Roles of the conserved tryptophan residues in transmembrane helix 6 in agonist-induced receptor internalization
We next examined whether mutations in the transmembrane domains of the receptors could influence the rate of receptor internalization. Our previous study showed that a highly conserved tryptophan residue (W279) in TM 6 of mR1 is critical for maintaining the conformation of mR1, and mutation of W279 to an alanine residue (R1-W279A) disturbed the hydrophobic interaction between TM5 and TM6, changing the receptor conformation and causing it to become constitutively active (57). We mutated the corresponding tryptophan in mR2 to an alanine (R2-W267A) and analyzed the internalization rate of this receptor mutant. In contrast to R1-W279A, which showed a more rapid internalization compared with mR1 (data not shown), R2-W267A displayed a slower internalization rate than mR2 (Fig. 4AGo, t1/2 = 8 ± 2 min for R2-W267A; t1/2 = 0.06 ± 0.05 min for R2, t1/2 = 1.0 ± 0.2 min for R1). We showed in a previous report that R1-W279A was basally active (57). The basal activities of R2-W267A were then compared with mR1 and mR2. Consistent with our previous observation, mR2 was constitutively active, whereas R2-W267A and mR1 were not basally active (Fig. 4BGo).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Effect of mutation of the conserved tryptophan in TM 6 on internalization and basal signaling activity of mR2. A, Transfected cells were incubated with 2 nM [3H]MeTRH at 37 C for the times indicated and specific and acid-resistant binding were measured as described in Materials and Methods. B, Cells were cotransfected with plasmid containing pFA2-CREB, pFR-Luc, and various amounts of plasmid encoding wild-type or R2-W267A and were assayed for luciferase activity as described in Materials and Methods. Results are expressed as mean ± SD of assays performed in triplicate in a representative experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chimeras of mR1 and mR2 were constructed and used in experiments to determine the structural basis of the different rates of agonist-induced receptor internalization. We demonstrated that the rate of agonist-induced internalization of a given chimera is more dependent on the origin of the extracellular domains and/or the transmembrane helices than on the origin of the intracellular domains of the receptors. These findings appear to contradict those of previous studies that have shown that the structural features of mR1 that determine the rate of agonist-induced internalization are located in the carboxyl terminus (6). However, these conclusions were based on studies using deletions of the carboxyl terminus of mR1. In the present study, we shifted the entire carboxyl termini of mR1 and mR2 to create chimeric receptors R1/R2-tail and R2/R1-tail that were shown to undergo agonist-induced internalization at a rate closely resembling mR1 and mR2, respectively. These data suggest that, although an intact carboxyl terminus is required for agonist-induced receptor internalization, it is not the structural feature(s) responsible for determining the internalization rate of mR1 and mR2; that is, other structural features may be more important in determining the rate of agonist-induced internalization in the presence of an intact carboxyl terminus. The conserved Trp of helix 6 in mR1 (R1-W279) had been shown in a previous study from our laboratory to form a hydrophobic cluster with amino acids of helix 5, and the interaction between helix 5 and 6 constrains the mR1 in an inactive conformation. Substitution of Trp 279 by Ala rendered mR1 constitutively active (57). The agonist-induced internalization rate of R1-W279A is 10-fold higher than that of wild-type mR1 (data not shown). In contrast, substitution of this conserved Trp with Ala in mR2 (R2-W267A) decreased both the basal signaling activity and the agonist-induced internalization rate. These findings indicated that the conserved Trp in helix 6 not only plays a role in determining the basal signaling activity of mR1 and mR2 but also greatly affects the rate of agonist-induced internalization of these receptors. In supporting our findings that the amino acids in the helical domains are important for determining basal activities and internalization rates of TRH Rs, we mutated G200 (TM5) and A252 (TM6) in mR2 to their corresponding amino acid in mR1, leucine, and valine, respectively, and the mutated receptors showed decreased basal activities and internalization rates (our unpublished observation). It is important to note that other structural features could affect agonist-induced internalization rates of TRH Rs. In fact, there is accumulating evidence indicating that the domains/residues involved in receptor internalization are multisite in nature. It is tempting to speculate that there is a specific conformation of GPCRs destined for internalization, just as there is an active conformation of GPCRs responsible for G protein activation and that these conformations are similar. Therefore, any mutation that facilities or impairs the conformational change leading to this specific state would affect receptor internalization and basal activity. Different domains may function independently or cooperatively in determining the rate of receptor internalization. It will be of interest to examine whether the mutation of the carboxyl terminus of mR1 could impair the high receptor internalization rate of mR2 in the chimeric receptor R2/R1-tail, or whether R1-W279A could rescue the slow internalization of the carboxyl-terminal deletion form of mR1.

TRH Rs that were only minimally basally active responded to TRH stimulation to a greater extent than constitutively active receptors. Relative refractoriness to further stimulation by agonist has also been reported for constitutively active forms of human LH Rs (31, 58). It is known that internalization of TRH is involved in the termination of its actions; therefore, the lack of responsiveness detected in cells expressing constitutively active receptors may be due in part to the rapid internalization of the TRH/TRH R complex. This would support the hypothesis that TRH R signaling occurs only when the active receptor is on the cell surface. In addition, the constitutively active receptors with rapid internalization rates might be down-regulated to a greater extent (59).

Although we have not found the explanation for the correlation between basal signaling activity and the rate of agonist-induced internalization, we suggest that a likely explanation is that the conformation of the active states of these receptors allows for more efficient coupling to G proteins and binding to the proteins involved in internalization. This is in agreement with the finding of several studies that have demonstrated that mutations that impair signaling of GPCRs reduce agonist-induced receptor internalization, whereas mutations that yield constitutively active receptors generate receptors that are internalized faster than wild-type receptors (21, 22, 31, 60). Another possibility is that active receptors, which are known to be posttranslationally modified, are more efficiently bound to the internalization machinery because they are modified. For example, phosphorylation of a number of GPCRs has been shown to be necessary for agonist-induced receptor internalization (12, 61). It is interesting to note that some constitutively active mutants of GPCRs showed a high level of basal phosphorylation (62, 63). It is possible, therefore, that the constitutively active TRH Rs are basally phosphorylated and interact with the internalization components in the ligand-free state and upon ligand binding more readily undergo agonist-induced internalization.


   Acknowledgments
 
We thank Wei Huang for discussions about mutagenesis strategies. We also thank Dr. Bruce Raaka for reading the manuscript.


   Footnotes
 
This work was supported in part by Grant DK-43031 from the National Institutes of Health.

Abbreviations: CREB, cAMP response element binding protein; EM, Epstein-Barr macrophage scavenger receptor; E/TM, extracellular and transmembrane; FSH R, FSH receptor; GnRH R, GnRH receptor; GPCRs, G protein-coupled receptors; I1, I2, or I3, first, second, and third intracellular loops; HEK, human embryonic kidney; IP, inositol phosphate; LH R, lutropin/choriogonadotropin receptor; mR1 or mR2, mouse TRH receptor type 1 or 2; R1 or R2, receptor type 1 or 2; TM 2 or 6, transmembrane helix 2 or 6; TRH, thyrotropin-releasing hormone; TRH R, TRH receptor.

Received February 5, 2002.

Accepted for publication April 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bunemann M, Hosey MM 1999 G-protein coupled receptor kinases as modulators of G-protein signalling. J Physiol 517(Part 1):5–23
  2. Carman CV, Benovic JL 1998 G-protein-coupled receptors: turn-ons and turn-offs. Curr Opin Neurobiol 8:335–344[CrossRef][Medline]
  3. Ferguson SS 2001 Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24[Abstract/Free Full Text]
  4. Lefkowitz RJ 1998 G protein-coupled receptors. III. New roles for receptor kinases and ß-arrestins in receptor signaling and desensitization. J Biol Chem 273:18677–18680[Free Full Text]
  5. Rodriguez MC, Xie YB, Wang H, Collison K, Segaloff DL 1992 Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization. Mol Endocrinol 6:327–336[Abstract/Free Full Text]
  6. Nussenzveig DR, Heinflink M, Gershengorn MC 1993 Agonist-stimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus. J Biol Chem 268:2389–2392[Abstract/Free Full Text]
  7. Gabilondo AM, Hegler J, Krasel C, Boivin-Jahns V, Hein L, Lohse MJ 1997 A dileucine motif in the C terminus of the ß2-adrenergic receptor is involved in receptor internalization. Proc Natl Acad Sci USA 94:12285–12290[Abstract/Free Full Text]
  8. Faussner A, Proud D, Towns M, Bathon JM 1998 Influence of the cytosolic carboxyl termini of human B1 and B2 kinin receptors on receptor sequestration, ligand internalization, and signal transduction. J Biol Chem 273:2617–2623[Abstract/Free Full Text]
  9. Parent JL, Labrecque P, Orsini MJ, Benovic JL 1999 Internalization of the TXA2 receptor {alpha} and ß isoforms. Role of the differentially spliced COOH terminus in agonist-promoted receptor internalization. J Biol Chem 274:8941–8948[Abstract/Free Full Text]
  10. Kishi M, Ascoli M 2000 The C-terminal tail of the rat lutropin/choriogonadotropin (CG) receptor independently modulates human (h)CG-induced internalization of the cell surface receptor and the lysosomal targeting of the internalized hCG-receptor complex. Mol Endocrinol 14:926–936[Abstract/Free Full Text]
  11. Kishi M, Liu X, Hirakawa T, Reczek D, Bretscher A, Ascoli M 2001 Identification of two distinct structural motifs that, when added to the c-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway. Mol Endocrinol 15:1624–1635[Abstract/Free Full Text]
  12. Lazari MF, Bertrand JE, Nakamura K, Liu X, Krupnick JG, Benovic JL, Ascoli M 1998 Mutation of individual serine residues in the C-terminal tail of the lutropin/choriogonadotropin receptor reveal distinct structural requirements for agonist-induced uncoupling and agonist-induced internalization. J Biol Chem 273:18316–18324[Abstract/Free Full Text]
  13. Arora KK, Sakai A, Catt KJ 1995 Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. J Biol Chem 270:22820–22826[Abstract/Free Full Text]
  14. Nakamura K, Liu X, Ascoli M 2000 Seven non-contiguous intracellular residues of the lutropin/choriogonadotropin receptor dictate the rate of agonist-induced internalization and its sensitivity to non-visual arrestins. J Biol Chem 275:241–247[Abstract/Free Full Text]
  15. Nakamura K, Liu X, Ascoli M 1999 The rate of internalization of the gonadotropin receptors is greatly affected by the origin of the extracellular domain. J Biol Chem 274:25426–25432[Abstract/Free Full Text]
  16. Li S, Liu X, Min L, Ascoli M 2001 Mutations of the second extracellular loop of the human lutropin receptor emphasize the importance of receptor activation and de-emphasize the importance of receptor phosphorylation in agonist-induced internalization. J Biol Chem 276:7968–7973[Abstract/Free Full Text]
  17. Kishi H, Ascoli M 2000 Multiple distant amino acid residues present in the serpentine region of the follitropin receptor modulate the rate of agonist-induced internalization. J Biol Chem 275:31030–31037[Abstract/Free Full Text]
  18. Hoelscher SR, Sairam MR, Ascoli M 1991 The slow rate of internalization of deglycosylated human chorionic gonadotropin is not due to its inability to stimulate cyclic adenosine monophosphate accumulation. Endocrinology 128:2837–2843[Abstract/Free Full Text]
  19. von Zastrow M, Kobilka BK 1994 Antagonist-dependent and -independent steps in the mechanism of adrenergic receptor internalization. J Biol Chem 269:18448–18452[Abstract/Free Full Text]
  20. Szekeres PG, Koenig JA, Edwardson JM 1998 The relationship between agonist intrinsic activity and the rate of endocytosis of muscarinic receptors in a human neuroblastoma cell line. Mol Pharmacol 53:759–765[Abstract/Free Full Text]
  21. Dhanwada KR, Vijapurkar U, Ascoli M 1996 Two mutations of the lutropin/choriogonadotropin receptor that impair signal transduction also interfere with receptor-mediated endocytosis. Mol Endocrinol 10:544–554[Abstract/Free Full Text]
  22. Barak LS, Menard L, Ferguson SS, Colapietro AM, Caron MG 1995 The conserved seven-transmembrane sequence NP(X)2, 3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the ß 2-adrenergic receptor. Biochemistry 34:15407–15414[CrossRef][Medline]
  23. Nussenzveig DR, Heinflink M, Gershengorn MC 1993 Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C. Mol Endocrinol 7:1105–1111[Abstract/Free Full Text]
  24. Cheung AH, Dixon RA, Hill WS, Sigal IS, Strader CD 1990 Separation of the structural requirements for agonist-promoted activation and sequestration of the ß-adrenergic receptor. Mol Pharmacol 37:775–977[Abstract]
  25. Hunyady L, Baukal AJ, Balla T, Catt KJ 1994 Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction. J Biol Chem 269:24798–24804[Abstract/Free Full Text]
  26. Conchon S, Monnot C, Teutsch B, Corvol P, Clauser E 1994 Internalization of the rat AT1a and AT1b receptors: pharmacological and functional requirements. FEBS Lett 349:365–370[CrossRef][Medline]
  27. Zanolari B, Raths S, Singer-Kruger B, Riezman H 1992 Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction. Cell 71:755–763[CrossRef][Medline]
  28. Clark RB, Friedman J, Prashad N, Ruoho AE 1985 Epinephrine-induced sequestration of the ß-adrenergic receptor in cultured S49 WT and cyc- lymphoma cells. J Cyclic Nucleotide Protein Phosphor Res 10:97–119[Medline]
  29. Yu R, Hinkle PM 1999 Signal transduction and hormone-dependent internalization of the thyrotropin-releasing hormone receptor in cells lacking Gq and G11. J Biol Chem 274:15745–15750[Abstract/Free Full Text]
  30. Petrou C, Chen L, Tashjian Jr AH 1997 A receptor-G protein coupling-independent step in the internalization of the thyrotropin-releasing hormone receptor. J Biol Chem 272:2326–2333[Abstract/Free Full Text]
  31. Min L, Ascoli M 2000 Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor. Mol Endocrinol 14:1797–1810[Abstract/Free Full Text]
  32. Min L, Galet C, Ascoli M 2001 The association of arrestin-3 with the human lutropin/choriogonadotropin receptor depends mostly on receptor activation rather than on receptor phosphorylation. J Biol Chem 277:702–710[Abstract/Free Full Text]
  33. Nakamura K, Krupnick JG, Benovic JL, Ascoli M 1998 Signaling and phosphorylation-impaired mutants of the rat follitropin receptor reveal an activation- and phosphorylation-independent but arrestin-dependent pathway for internalization. J Biol Chem 273:24346–24354[Abstract/Free Full Text]
  34. Gershengorn MC, Osman R 1996 Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev 76:175–191[Abstract/Free Full Text]
  35. Aragay AM, Katz A, Simon MI 1992 The G {alpha} q and G {alpha} 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells. J Biol Chem 267:24983–24988[Abstract/Free Full Text]
  36. Gershengorn MC 1989 Mechanism of signal transduction by TRH. Ann NY Acad Sci 553:191–196[CrossRef][Medline]
  37. Hsieh KP, Martin TF 1992 Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. Mol Endocrinol 6:1673–1681[Abstract/Free Full Text]
  38. Ashworth R, Yu R, Nelson EJ, Dermer S, Gershengorn MC, Hinkle PM 1995 Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling. Proc Natl Acad Sci USA 92:512–516[Abstract/Free Full Text]
  39. Yu R, Ashworth R, Hinkle PM 1998 Receptors for thyrotropin-releasing hormone on rat lactotropes and thyrotropes. Thyroid 8:887–894[Medline]
  40. Buck F, Wang W, Harder S, Brathwaite C, Bruhn TO, Gershengorn MC 2000 Juxtamembrane regions in the third intracellular loop of the thyrotropin-releasing hormone receptor type 1 are important for coupling to Gq. Endocrinology 141:3717–3722[Abstract/Free Full Text]
  41. Straub RE, Frech GC, Joho RH, Gershengorn MC 1990 Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc Natl Acad Sci USA 87:9514–9518[Abstract/Free Full Text]
  42. Zhao D, Yang J, Jones KE, Gerald C, Suzuki Y, Hogan PG, Chin WW, Tashjian Jr AH 1992 Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells. Endocrinology 130:3529–3536[Abstract/Free Full Text]
  43. Sellar RE, Taylor PL, Lamb RF, Zabavnik J, Anderson L, Eidne KA 1993 Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. J Mol Endocrinol 10:199–206[Abstract/Free Full Text]
  44. de la Pena P, Delgado LM, del Camino D, Barros F 1992 Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells. Biochem J 284(Part 3):891–899
  45. Duthie SM, Taylor PL, Anderson L, Cook J, Eidne KA 1993 Cloning and functional characterisation of the human TRH receptor. Mol Cell Endocrinol 95:R11–R15
  46. Matre V, Karlsen HE, Wright MS, Lundell I, Fjeldheim AK, Gabrielsen OS, Larhammar D, Gautvik KM 1993 Molecular cloning of a functional human thyrotropin-releasing hormone receptor. Biochem Biophys Res Commun 195:179–185[CrossRef][Medline]
  47. Sun YM, Millar RP, Ho H, Gershengorn MC, Illing N 1998 Cloning and characterization of the chicken thyrotropin-releasing hormone receptor. Endocrinology 139:3390–3398[Abstract/Free Full Text]
  48. Takata M, Shimada Y, Ikeda A, Sekikawa K 1998 Molecular cloning of bovine thyrotropin-releasing hormone receptor gene. J Vet Med Sci 60:123–127[CrossRef][Medline]
  49. Itadani H, Nakamura T, Itoh J, Iwaasa H, Kanatani A, Borkowski J, Ihara M, Ohta M 1998 Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochem Biophys Res Commun 250:68–71[CrossRef][Medline]
  50. Cao J, O’Donnell D, Vu H, Payza K, Pou C, Godbout C, Jakob A, Pelletier M, Lembo P, Ahmad S, Walker P 1998 Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J Biol Chem 273:32281–32287[Abstract/Free Full Text]
  51. O’Dowd BF, Lee DK, Huang W, Nguyen T, Cheng R, Liu Y, Wang B, Gershengorn MC, George SR 2000 TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Mol Endocrinol 14:183–193[Abstract/Free Full Text]
  52. Harder S, Lu X, Wang W, Buck F, Gershengorn MC, Bruhn TO 2001 Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2). Endocrinology 142:1188–1194[Abstract/Free Full Text]
  53. Wang W, Gershengorn MC 1999 Rat TRH receptor type 2 exhibits higher basal signaling activity than TRH receptor type 1. Endocrinology 140:4916–4919[Abstract/Free Full Text]
  54. Nussenzveig DR, Thaw CN, Gershengorn MC 1994 Inhibition of inositol phosphate second messenger formation by intracellular loop one of a human calcitonin receptor. Expression and mutational analysis of synthetic receptor genes. J Biol Chem 269:28123–28129[Abstract/Free Full Text]
  55. Perlman JH, Colson AO, Wang W, Bence K, Osman R, Gershengorn MC 1997 Interactions between conserved residues in transmembrane helices 1, 2, and 7 of the thyrotropin-releasing hormone receptor. J Biol Chem 272:11937–11942[Abstract/Free Full Text]
  56. Jinsi-Parimoo A, Gershengorn MC 1997 Constitutive activity of native thyrotropin-releasing hormone receptors revealed using a protein kinase C-responsive reporter gene. Endocrinology 138:1471–1475[Abstract/Free Full Text]
  57. Colson AO, Perlman JH, Jinsi-Parimoo A, Nussenzveig DR, Osman R, Gershengorn MC 1998 A hydrophobic cluster between transmembrane helices 5 and 6 constrains the thyrotropin-releasing hormone receptor in an inactive conformation. Mol Pharmacol 54:968–978[Abstract/Free Full Text]
  58. Shinozaki H, Fanelli F, Liu X, Jaquette J, Nakamura K, Segaloff DL 2001 Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness. Mol Endocrinol 15:972–984[Abstract/Free Full Text]
  59. Nakamura K, Lazari MF, Li S, Korgaonkar C, Ascoli M 1999 Role of the rate of internalization of the agonist-receptor complex on the agonist-induced down-regulation of the lutropin/choriogonadotropin receptor. Mol Endocrinol 13:1295–1304[Abstract/Free Full Text]
  60. Min KS, Liu X, Fabritz J, Jaquette J, Abell AN, Ascoli M 1998 Mutations that induce constitutive activation and mutations that impair signal transduction modulate the basal and/or agonist-stimulated internalization of the lutropin/choriogonadotropin receptor. J Biol Chem 273:34911–34919[Abstract/Free Full Text]
  61. Menard L, Ferguson SS, Zhang J, Lin FT, Lefkowitz RJ, Caron MG, Barak LS 1997 Synergistic regulation of ß2-adrenergic receptor sequestration: intracellular complement of ß-adrenergic receptor kinase and ß-arrestin determine kinetics of internalization. Mol Pharmacol 51:800–808[Abstract/Free Full Text]
  62. Ren Q, Kurose H, Lefkowitz RJ, Cotecchia S 1993 Constitutively active mutants of the {alpha} 2-adrenergic receptor. J Biol Chem 268:16483–16487[Abstract/Free Full Text]
  63. Westphal RS, Backstrom JR, Sanders-Bush E 1995 Increased basal phosphorylation of the constitutively active serotonin 2C receptor accompanies agonist-mediated desensitization. Mol Pharmacol 48:200–205[Abstract]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Engel, S. Neumann, N. Kaur, V. Monga, R. Jain, J. Northup, and M. C. Gershengorn
Low Affinity Analogs of Thyrotropin-releasing Hormone Are Super-agonists
J. Biol. Chem., May 12, 2006; 281(19): 13103 - 13109.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
D. Du, B. M. Raaka, H. Grimberg, M. Lupu-Meiri, Y. Oron, and M. C. Gershengorn
Carboxyl Tail Cysteine Mutants of the Thyrotropin-Releasing Hormone Receptor Type 1 Exhibit Constitutive Signaling: Role of Palmitoylation
Mol. Pharmacol., July 1, 2005; 68(1): 204 - 209.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
X. Lu, W. Huang, S. Worthington, P. Drabik, R. Osman, and M. C. Gershengorn
A Model of Inverse Agonist Action at Thyrotropin-Releasing Hormone Receptor Type 1: Role of a Conserved Tryptophan in Helix 6
Mol. Pharmacol., November 1, 2004; 66(5): 1192 - 1200.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. C. Hanyaloglu, R. M. Seeber, T. A. Kohout, R. J. Lefkowitz, and K. A. Eidne
Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes. DIFFERENTIAL REGULATION OF beta -ARRESTINS 1 AND 2
J. Biol. Chem., December 20, 2002; 277(52): 50422 - 50430.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sun, Y.
Right arrow Articles by Gershengorn, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sun, Y.
Right arrow Articles by Gershengorn, M. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals