Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement.

Congenital central hypothyroidism occurs either in isolation or in conjunction with other pituitary hormone deficits. Using exome and candidate gene sequencing, we identified 8 distinct mutations and 2 deletions in IGSF1 in males from 11 unrelated families with central hypothyroidism, testicular enlargement and variably low prolactin concentrations. IGSF1 is a membrane glycoprotein that is highly expressed in the anterior pituitary gland, and the identified mutations impair its trafficking to the cell surface in heterologous cells. Igsf1-deficient male mice show diminished pituitary and serum thyroid-stimulating hormone (TSH) concentrations, reduced pituitary thyrotropin-releasing hormone (TRH) receptor expression, decreased triiodothyronine concentrations and increased body mass. Collectively, our observations delineate a new X-linked disorder in which loss-of-function mutations in IGSF1 cause central hypothyroidism, likely secondary to an associated impairment in pituitary TRH signaling.

Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization.

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.

Lithium modulates expression of TRH receptors and TRH-related peptides in rat brain.

Lithium is an established mood stabilizer and neuroprotective agent frequently used in the treatment of bipolar disorder and as an adjuvant in drug-resistant unipolar depression. The mechanisms underlying both the therapeutic efficacy of lithium and the exacerbation of symptoms following rapid withdrawal are not understood. From previous studies showing antidepressant and neuroprotective activities of thyrotropin releasing hormone (TRH) and TRH-related neuropeptides we hypothesized that lithium may have substantial effects on the expression and secretion of these peptides and/or their receptors in various rat brain regions involved in the regulation of mood. Chronic lithium effect on TRH receptor binding studies: The effect of 1 and 2 weeks of dietary lithium on [(3)H]3-Me-His-TRH binding to plasma membranes of nucleus accumbens, amygdala and pituitary of young adult male Wistar and the endogenously 'depressed' Wistar Kyoto (WKY) rats was measured by the method of Burt and Taylor [Burt, D.R., Taylor, R.L., Endocrinology 106 (1980) 1416-1423]. Acute, chronic and withdrawal effect of lithium on TRH and TRH-like peptide levels in young, adult male Sprague-Dawley rats: Rats were divided into four lithium treatment groups. Control animals received a standard laboratory rodent chow. The acute group received a single i.p. injection of 1.5 milli-equivalents of LiCl 2 h prior to killing. The chronic and withdrawal groups received standard rodent chow containing 1.7 g/kg LiCl for 2 weeks. Withdrawal rats were returned to standard chow 48 h prior to killing while the chronic animals continued on the LiCl diet. TRH, TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP (pGlu-Glu-Pro-NH(2), a TRH-like peptide with antidepressant activity) and Ps4 (a prepro-TRH-derived TRH-enhancing decapeptide) immunoreactivity (IR) were measured in 13 brain regions. The remaining samples were pooled and fractionated by high-pressure liquid chromatography followed by EEP radioimmunoassay. Chronic lithium treatment increased [(3)H]3Me-TRH binding in the nucleus accumbens and amygdala about two-fold in both Wistar and WKY rats but no change was observed in pituitary binding. The most widespread changes in TRH and TRH-related peptide levels were observed in the withdrawal group compared to the controls. The direction of change for the total IR was consistent for all TRH-IR and TRH-related peptide-IR within a given tissue. For example, withdrawal increased all peptide levels in the pyriform cortex and striatum but decreased these levels in the anterior cingulate and lateral cerebellum. Both acute injection and chronic treatment with LiCl decreased TRH and TRH-related peptide levels in the entorhinal cortex. Acute injection and withdrawal both increased EEP-IR in striatum by more than two-fold. The acute effects are most likely due to changes in the release of these peptides since 2 h is not sufficient time for alterations in peptide biosynthesis. Chronic treatment increased levels of pGlu-Phe-Pro-NH(2) levels in hippocampus, pGlu-Leu-Pro-NH(2), and peak '2' in septum by more than four-fold. The present results are consistent with a component role for TRH and related peptides in the mood-altering effects of lithium administration and withdrawal frequently observed during treatment for depression and bipolar disorder.

[Expression of thyrotropin-releasing hormone receptor mRNA in the rat testis development].

In order to investigate the regulation of thyrotrophin-releasing hormone receptor (TRH-R) expression in rat testis, and to study their function in spermatogenesis, oligonucleotide primers were designed from the sequences of rat pituitary TRH-R cDNA for reverse transcription-polymerase chain reaction (RT-PCR). Specific fragments of TRH-R cDNA were cloned. DNA sequence analysis indicated that cDNA sequence of TRH-R from rat testis was consistent with those of pituitary TRH-R cDNA. The non-radioactive in situ hybridization (NR-ISH) technique was applied to localize cells encoding TRH-R mRNA in the rat testis. Hybridization signals were detected exclusively in the leydig cells, but not in the spermatogenetic cells of the rat testis. TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats at different developmental stages by real time quantitative RT-PCR. The quantitative analyses demonstrated that no TRH mRNA could be detected at the earliest stage (day 8). TRH mRNA signals were detected on day 15 and increased progressively on day 20, 35, 60 and 90. These results suggested that rat testis could specifically express TRH-R, and the transcription of TRH-R gene in the rat testis was development-dependent.

Expression of thyrotropin-releasing hormone (TRH) receptor subtype 1 in mouse pancreatic islets and HIT-T15, an insulin-secreting clonal beta cell line.

Thyrotropin-releasing hormone (TRH), originally isolated as a hypothalamic hormone, has been reported to be present and released from the pancreatic beta cells, affecting pancreatic functions. However, it still remains unclear whether TRH receptor is expressed in the pancreas. In the present study, we characterized TRH receptors (TRHR) in mouse pancreatic islets and HIT-T15 cells, a hamster clonal beta cell line. RT-PCR study showed significant expression of TRHR subtype 1 (TRHR1) mRNA in both mouse pancreatic islets and HIT-T15 (HIT) cells. In contrast, there was no expression of TRHR2 mRNA, a novel subtype of TRHR which is expressed predominantly in the central nervous system. Sequencing analysis demonstrated that TRHR1 of the islets was identical to that in the pituitary, and cloned hamster TRHR1 shared 93.3 % homology with that of the mouse at the nucleic acid level. Northern blot analysis of TRHR 1 mRNA in HIT-T15 cells showed a single strong hybridization signal approximately 3.7 kb in length. Furthermore, Scatchard plot analysis in HIT-T15 cells revealed that the Kd value for MeTRH was 0.63 nM. Significant elevation of intracellular calcium concentration was observed in response to as little as 10 nM TRH , and this was not affected by removal of extracellular calcium. This is the first description indicating the presence of functional TRH receptor subtype 1 in the pancreatic beta cells, and our observations suggested the regulation of pancreatic function by TRH through autocrine or paracrine mechanisms.

Effects of pilocarpine- and kainate-induced seizures on thyrotropin-releasing hormone biosynthesis and receptors in the rat brain.

The expression of mRNA coding for prepro-thyrotropin releasing hormone (preproTRH) was estimated in the rat brain in two animal models of limbic seizures, evoked by systemic administration of pilocarpine (400 mg/kg ip) or kainate (12 mg/kg ip). As shown by an in situ hybridization study, after 24h both pilocarpine- and kainate-induced seizures profoundly increased the preproTRH mRNA level in the dentate gyrus. After 72h, the preproTRH mRNA level was back to control values. Kainate-treated rats showed an elevated level of TRH in the hippocampus, septum, frontal and occipital cortex after 24 and 72h, whereas in the striatum and amygdala the TRH level was raised after 72h only. In the hypothalamus, TRH levels was lowered after 3 and 24h, and returned to the control after 72h. Pilocarpine-induced seizures also elevated the TRH level after 72h in the majority of the above structures, except for the hypothalamus and amygdala where no changes were found at any time point. A radioreceptor assay showed that kainate decreased the Bmax value of TRH receptors in the striatum and hippocampus after 3 and 24h, respectively, and had no effect on the Kd values. In contrast, pilocarpine-induced seizures lowered the Bmax of TRH receptors in the striatum, hippocampus and piriform cortex after 72h only, and decreased Kd values in the striatum, amygdala and frontal cortex. These data showed that pilocarpine- and kainate-induced seizures enhanced likewise preproTRH mRNA in the dentate gyrus; on the other hand, they differed with respect to time- and structure-related changes in TRH tissue levels and TRH receptors. These differences may have functional significance in TRH-dependent control mechanism of the seizure activity in these two models of limbic epilepsy.

Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor.

The long isoform of the rat thyrotropin-releasing hormone receptor (TRHR) was modified by the addition of a vesicular stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP). VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified receptor and stimulated [3H]inositol phosphate production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular exposure to TRH. Internalization was detected within 3-5 min after treatment with 1 x 10(-7) M TRH, with dramatic reductions in plasma membrane localization achieved within 10-15 min. The TRHR antagonist/inverse agonist chlordiazepoxide competitively inhibited internalization. Hyperosmotic sucrose inhibited internalization of VSV-TRHR-GFP, measured both by intact cell [3H]TRH binding studies and by confocal microscopy. Now TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but plasma membrane-delineated foci. Pretreatment with the microtubule-disrupting agent nocodazole allowed internalization of the VSV-TRHR-GFP construct but only into vesicles that remained in close apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and Texas Red transferrin initially indicated entirely separate localizations. After exposure to TRH substantial amounts of VSV-TRHR-GFP were present in vesicles overlapping those containing Texas Red transferrin. Such results demonstrate the G protein-coupling capacity and provide real time visualization of the processes of internalization of a TRH-receptor-GFP construct in response to agonist.

Molecular cloning of bovine thyrotropin-releasing hormone receptor gene.

The genomic DNA encoding bovine Thyrotropin-Releasing Hormone Receptor (TRHR) was isolated from a bovine (Holstein) genomic library. Using PCR fragments of bovine candidate TRHR Transmembrane domain (TMD-III-IV) and C-terminus domain of mouse TRHR cDNA as probes, 9 x 10(5) plaques were screened to obtain several clones each containing the N-terminus or C-terminus domain. The bovine TRHR gene encoded 398 amino acids and has a long intron. The identity of the deduced amino acid sequence of bovine TRHR exceeded 88% that of mouse, rat or human. RT-PCR analysis indicated TRHR mRNA to be expressed in the pituitary and brain.

The detection of thyrotropin-releasing hormone (TRH) and TRH receptor gene expression in Siberian hamster testes.

Thyrotropin-releasing hormone (TRH) from the hypothalamus is the major regulator of TSH synthesis and secretion. Most recently, TRH and TRH receptors (TRH-R), as well as their mRNAs, have been identified in rat testis. To expand our knowledge on the testicular TRH and TRH receptor gene expression in different species, in the present study the mRNA levels of testicular TRH and TRH-R were investigated in Siberian hamsters. To further localize the cellular sites of the gene expression, the animal model was treated with a single injection of ethylene dimethane sulfonate (EDS) (i.p., 80 mg/kg body weight), a compound known as to specifically eliminate testicular Leydig cells. The elimination of Leydig cells induced by EDS treatment was confirmed by histological studies of the testis sections and by serum hormonal analyses, which showed a dramatic reduction of serum testosterone (T) levels and significantly elevated serum LH concentrations. Messenger RNA levels of TRH and TRH-R in the testes were determined by Northern blot analyses quantitated with densitometry scanning. The results showed that specific TRH-R mRNA, 3.8 kb in size, was identified in Siberian hamster testes and the mRNA levels were significantly elevated in the EDS-treated testes compared to the controls (p < 0.01). Testicular TRH mRNA was also detected; however, no significant differences in TRH mRNA levels were found between EDS-treated and control groups. The size of TRH mRNA was characterized as about 1.2 kb in hamster testes, which was smaller than that observed in the rat hypothalamus (1.6 kb) and in the rat testis (2.0 kb). Further studies by RNase H digestion revealed the presence of smaller TRH transcripts in the hamster testes than those in the rat testis. No hybridization signal for TRH mRNA was detected by RNase protection assay, when a rat TRH riboprobe was applied to hamster testis RNA, suggesting the limited homology of TRH gene sequences between these two species. Our results demonstrate that both TRH and TRH-R genes are expressed in Siberian hamster testes, and a significant increase of TRH-R mRNA levels occurs in the Leydig cell eliminated hamster testes. Unlike the rat testicular TRH mRNA mainly detected in Leydig cells, in hamster TRH mRNA could also be detected in other testicular compartment.

Ectopic expression of thyrotropin releasing hormone (TRH) receptors in liver modulates organ function to regulate blood glucose by TRH.

Maintenance of blood glucose by the liver is normally initiated by extracellular regulatory molecules such as glucagon and vasopressin triggering specific hepatocyte receptors to activate the cAMP or phosphoinositide signal transduction pathways, respectively. We now show that the normal ligand-receptor regulators of blood glucose in the liver can be bypassed using an adenovirus vector expressing the mouse pituitary thyrotropin releasing hormone receptor (TRHR) cDNA ectopically in rat liver in vivo. The ectopically expressed TRHR links to the phosphoinositide pathway, providing a means to regulate liver function with TRH, an extracellular ligand that does not normally affect hepatic function. Administration of TRH to these animals activates the phosphoinositide pathway, resulting in a sustained rise in blood glucose. It should be possible to use this general strategy to modulate the differentiated functions of target organs in a wide variety of pathologic states.

Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling.

Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins.

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Identification of Asn289 as a ligand binding site in the rat thyrotropin-releasing hormone (THR) receptor as determined by complementary modifications in the ligand and receptor: a new model for THR binding.

To test the hypothesis that pGlu of the thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2) binds to Asn289 in the third extracellular loop (EL3) of its receptor through a hydrogen bonding interaction, we converted Asn289 to Asp (N289D mutant) and measured the potencies of TRH and Pro1TRH for the wild-type and mutant receptors. TRH was 100 times less potent for the N289D receptor than for the wild-type. In contrast, Pro1TRH, which has a protonated proline in place of the pGlu of TRH, was 10 times more potent for the N289D receptor than for the wild-type. A similar result was obtained when Asn289 was converted to Glu, while the potency of Pro1TRH did not change when Asn289 was converted to Ala, confirming that the increased potency of Pro1TRH for the N289D receptor was due to a charge interaction between Pro1TRH and the mutant receptor. These findings are inconsistent with a previous model indicating a direct interaction of the pGlu of TRH with Asn110 in the third transmembrane helix of the receptor (Perlman et al. (1994) J. Biol. Chem. 269, 23383-23386). When Asn110 was converted to Asp (N110D mutant), unlike the N289D receptor, the potency of Pro1TRH for the N110D receptor was decreased by > 10-fold rather than increased. Therefore, a direct interaction of Asn110 with the pGlu of TRH could not be supported by our experiments. We propose a new model in which the pGlu of TRH binds to Asn289 in EL3 and conclude that, unlike catecholamines which bind completely within the transmembrane domain of their receptors, this tripeptide binds, at least in part, to the extracellular domain of its receptor.

TRH receptor on immune cells: in vitro and in vivo stimulation of human lymphocyte and rat splenocyte DNA synthesis by TRH.

This work examined whether (1) immune cells express thyrotrophin releasing hormone (TRH) receptor mRNA and (2) TRH modulates lymphocyte activation. By Northern blot of RNA extracted from human peripheral blood mononuclear cells (PBMC) and rat splenocytes, a single TRH receptor mRNA band of about 3.8 kb (identical to that obtained from pituitary cells) was obtained, under both basal and stimulated conditions. A significant increase in DNA synthesis was observed in phytohemagglutinin-stimulated PBMC and concanavalin A (Con A) stimulated splenocytes when TRH (10(-6) M-10(-12) M) was added. After 5, 30, 60, 180 min and 24 h of TRH administration in vivo, a significant increase in the rat splenocyte proliferative response to Con A was observed. In vivo administration of anti-rat TSH antibody (1/1000) blocked the increase observed after 30 min of TRH administration on the Con A stimulated splenocyte response. TRH possess immunostimulatory functions directly via its receptor and indirectly via release of other immunostimulatory factors such as thyrotrophin.

Desensitization of the response to thyrotropin-releasing hormone in Xenopus oocytes is an amplified process that precedes calcium mobilization.

Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3 - 15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of co-expressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by beta-phorbol 12-myristate, 13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C consensus phosphorylation sites in the C-terminal region, exhibited desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

The hemispheric functional expression of the thyrotropin-releasing-hormone receptor is not determined by the receptors' physical distribution.

The thyrotropin-releasing-hormone receptor (TRH-R) is a member of a family of the G-protein-coupled receptors that share structural similarities and exert their physiological action via the inositol lipid signal-transduction pathway. The TRH-R when expressed in Xenopus oocytes exhibits marked preference of the response (increased chloride conductance) for the animal hemisphere. Whereas the rat TRH-R functional distribution was strongly asymmetric (animal/vegetal ratio = 9.5), the mouse TRH-R exhibited a significantly lower ratio (3.9). Truncation of the last 59 amino acids of the C-terminal region of the mouse TRH-R did not lead to any changes in the functional hemispheric distribution. Despite the polarization of response, receptor number was similar on both hemispheres. Moreover, the apparent half-life of the functional expression of the TRH-R was approx. 4 h on both hemispheres when the expression was inhibited by a specific antisense oligonucleotide. Inhibition of total protein synthesis with cycloheximide affected hemispheric responses mediated by each of the three TRH-Rs tested in a qualitatively different way. These results suggest that an additional, rapidly degraded, protein modulates the functional hemispheric expression of the TRH-Rs.

Thyrotropin-releasing hormone (TRH) receptor number determines the size of the TRH-responsive phosphoinositide pool. Demonstration using controlled expression of TRH receptors by adenovirus mediated gene transfer.

We use an adenovirus vector, AdCMVmTRHR, to express thyrotropin-releasing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is directly related to receptor number. Infection of HeLa cells with increasing numbers of AdCMVmTRHR caused time-dependent graded expression of TRH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permitted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in > or = 90% of HeLa cells. Measurement of prelabeled phosphoinositides hydrolyzed during prolonged TRH stimulation assesses the size of the TRH-responsive pool. In cells infected with AdCMVmTRHR for 24 h, the size of the TRH-responsive phosphoinositide pool increased with increasing TRH-R expression. The TRH-responsive pool also increased with time after infection as the number of TRH-Rs increased. Similar observations were made in GHY and KB cells. These data confirm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershengorn, M. C. (1990) Biochem. J. 271, 331-336) that there are hormone-responsive and -unresponsive pools of cellular phosphoinositides and that the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.

Differential effects of cytoskeletal agents on hemispheric functional expression of cell membrane receptors in Xenopus oocytes.

1. We studied the effects of three cytoskeleton-disrupting agents, colchicine (COL), vinblastine (VIN), cytochalasins, on the functional hemispheric expression of native muscarinic and acquired thyrotropin-releasing hormone receptors TRH-Rs). Responses in oocytes of common donors, which express M3-like receptors (M3Rs), were not affected by either COL or VIN on the animal hemisphere. The functional expression of M3Rs on the vegetal hemisphere was inhibited by 50%. Cytochalasin B caused a uniform inhibition (by 31-33%) of receptor functional expression on either hemisphere. 2. Oocytes of variant donors express predominantly M1-like receptors (M1Rs) on the animal and M3Rs on the vegetal hemisphere. In these oocytes, both COL and VIN caused approximately 50% inhibition of functional expression on either hemisphere. Cytochalasin B caused more extensive, though variable inhibition on both hemispheres. Both antitubulin agents had no effect on the functional expression of the TRH-Rs on either hemisphere. Cytochalasin B, however, caused an extensive inhibition of the functional expression of this receptor (by 70-75%). 3. Induction of maturation of oocytes (7-hr incubation with progesterone) resulted in a 66% decrease in the response to TRH, reflecting mainly a decrease on the animal hemisphere. Maturation in the presence of colchicine had no further effect on the activity measured on the animal hemisphere but caused a major increase in the activity on the vegetal hemisphere. This resulted in a dramatic change in animal/vegetal activity ratio (4.8 +/- 1.5 to 0.8 +/- 0.2). 4. It appears that while antitubulin drugs affect the functional expression of the three receptors at the two hemispheres differently, disruption of the microfilaments interferes uniformly with receptor functional expression. We suggest that microfilaments may be involved in a common component of the signal transduction pathway in oocytes or in the anchoring of receptors coupled to the guaninine nucleotide-binding regulatory proteins. Moreover, progesterone-induced changes in the functional organization of the signal transduction pathway appear to be controlled to a large extent by the tubulin component of the cytoskeleton.